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Cloning of and genetic variation in protease VCP1 from the nematophagous fungus Pochonia chlamydosporia
Abstract
The fungus Pochonia chlamydosporia is a biocontrol agent with commercial potential for root knot and cyst nematodes. It produces an alkaline serine protease, VCP1, during infection of nematode eggs. The gene encoding VCP1 was sequenced and the sequences of cDNAs from six isolates from different nematode hosts were compared. The gene encoding VCP1 was similar to PR1 from Metarhizium anisopliae with similar regulatory elements. Comparison of translated cDNA sequences revealed two amino acid polymorphisms at positions 65 and 99, indicating a difference between isolates from cyst and root nematodes. The positions and nature of the polymorphisms indicated that the two forms of VCP1 might have different properties and this was tested with five chromogenic polypeptide substrates. Enzyme assays revealed the two forms differed in their abilities to utilise Succ-Ala-Ala-Pro-Phe-pNa and Succ-Ala-Val-Pro-Phe-pNa, suggesting different amino acid affinities at the S3 binding region. This indicates host related genetic variation in VCP1 between isolates of P. chlamydosporia isolated from different nematode hosts, which might contribute to host preference. Such differences may be important in future exploitation of P. chlamydosporia as a nematode biocontrol agent.
Supplementary resources (6)
... Mendoza et al. (2013), observó que T. atroviride, T. harzianum y T. viride destruyen los huevos de Meloidogyne sp., en una secuencia parasítica que lleva a la destrucción completa en 72 horas. Respecto al hongo Pochonia chlamydosporia es un controlador biológico con potencial comercial para los nematodos del nudo y el quiste de la raíz; produce una serina proteasa alcalina, VCP1, durante la infección de huevos de nematodos (Morton et al., 2003); asimismo Luambano et al. (2015) mencionan que la eficacia de muchos agentes de control biológico, incluido P. chlamydosporia, depende de las condiciones del suelo. ...
... Se deduce que una variación genética relacionada con el huésped en VCP1 entre aislamientos de P. chlamydosporia aislados de diferentes huéspedes nematodos, lo que podría contribuir a la preferencia del huésped; tales diferencias pueden ser importantes en la explotación futura del hongo como agente de biocontrol de nematodos (Morton et al., 2003). La alta densidad de inóculo de nematodos resulta en una muerte considerable de la planta de tomate, lo que demuestra que el hongo no pudo controlar los altos niveles de nematodos; en la cosecha de la mayoría de los ciclos de cultivo, se encontraron menos J2 en el suelo o las raíces o se contaron menos masas de huevos por sistema de raíces en macetas con P. chlamydosporia en comparación con macetas sin el hongo; mientras una aplicación única de este hongo pudo ralentizar la acumulación de la población de M. javanica durante al menos 5 a 7 meses (Hoedekie et al., 2005). ...
... Cuando aumenta la dosis de aplicación de P. lilacinus y P. chlamydosporia disminuyen todas las variables a los 90 días del trasplante, como el número de nudos/planta, incidencia de nematodos/planta, severidad, número de huevos/planta, población de juveniles 2/nudo, población de número de hembras/nudo; mientras que con a medida que aumenta la dosis baja el nivel de todas las variables; mientras que para T. harzianum permanecen constantes todas las variables, lo que indica que no tiene efecto en el M. exigua. Tal como menciona sobre la incidencia (Puertas et al., 2006) hay efecto del hongo en la incidencia del nematodo; Morton et al. (2003) dice que el efecto del biocontrol que realiza P. chlamidosporia reduce la severidad del nematodo. Esto se atribuye a que Pochonia chlamydosporia presenta mayor eficiencia en el control de Meloidogyne exigua; así como lo mencionan Hoedekie et al. (2005), el efecto de control de este biocontrolador sobre la especie de M. exigua. ...
La selva central del Perú es la región más importante de producción de café a nivel nacional, donde observa una alta incidencia de nemátodo formador de agallas en las raíces del cafeto; Meloidogyne exigua Goeldi, no tiene un control adecuado ya que el control químico afecta la salud y el medio ambiente, en ese sentido se busca alternativas de control biológico. El objetivo fue probar la eficacia de tres hongos nematófagos contra M. exigua Goeldi utilizando plantas de vivero. El trabajo se realizó en laboratorio y campo. Las variables evaluadas fueron: número de agallas, incidencia de nematodo, severidad, población de huevos, población de juveniles 2 y población de hembras. Los resultados muestran que 1a aplicación de Pochonia chlamidosporia supera estadísticamente en el control de nematodos respecto a Paecilomyces lilacinus y Trichoderma harzianum, que ocupan el segundo y tercer lugar respectivamente. La dosis de 2 g/plántula para P. chlamidosporia supera estadísticamente a las dosis de 1,0 y 0,5 g/plántula. Cuando aumenta la dosis de aplicación de Paecilomyces lilacinus y Pochonia chlamydosporia disminuyen todas las variables; pero cuando aumenta la dosis de Trichoderma harzianum las variables permanecen constantes, por lo que no tiene efecto de control.
... Distinct variants of P. chlamydosporia are associated with different host nematode species, and strains isolated from root-knot nematodes and cyst nematodes do not anastomose; they are considered different biotypes 1 . The sequence of the VCP1 subtilisin encoding gene can be used to identify the biotypes of P. chlamydosporia 18 . The genetic variations between isolates from cyst and root-knot nematodes were also examined by other molecular methods 19,20 and demonstrated by virulence assay in vitro 21 . ...
... It had been deposited into the China General Microbiological Culture Collection Center (CGMCC, number 8860) 46 . Previously, the biotypes of P. chlamydosporia were identified by molecular methods [18][19][20] and in vitro assays that were also confirm by the detection of VCP1 gene sequences 21 . The variant sites of amino acid (aa) sequences of the VCP1 genes (subtilisins) 18 include two characteristic sites 171 and 208 ("E" and "G" in VCP1 of root-knot nematode isolates, and "Q" and "A" in VCP1 of cyst nematode isolates). ...
... Previously, the biotypes of P. chlamydosporia were identified by molecular methods [18][19][20] and in vitro assays that were also confirm by the detection of VCP1 gene sequences 21 . The variant sites of amino acid (aa) sequences of the VCP1 genes (subtilisins) 18 include two characteristic sites 171 and 208 ("E" and "G" in VCP1 of root-knot nematode isolates, and "Q" and "A" in VCP1 of cyst nematode isolates). For PC170, its infection ability to M. incognita eggs had been previously tested 36 and its previously sequenced (by PCR technology) beta-tubulin sequences (the same sequences of VFPPC_01610 gene in the assembled genome, from 953 bp to 1188 bp) are the most similar to the deposited AJ012713 sequences in NCBI collected data, which was from the P. chlamydosporia strain Vc10 isolated from M. incognita eggs in UK 58 . ...
Pochonia chlamydosporia infects eggs and females of economically important plant-parasitic nematodes. The fungal isolates parasitizing different nematodes are genetically distinct. To understand their intraspecific genetic differentiation, parasitic mechanisms, and adaptive evolution, we assembled seven putative chromosomes of P. chlamydosporia strain 170 isolated from root-knot nematode eggs (~44 Mb, including 7.19% of transposable elements) and compared them with the genome of the strain 123 (~41 Mb) isolated from cereal cyst nematode. We focus on secretomes of the fungus, which play important roles in pathogenicity and fungus-host/environment interactions, and identified 1,750 secreted proteins, with a high proportion of carboxypeptidases, subtilisins, and chitinases. We analyzed the phylogenies of these genes and predicted new pathogenic molecules. By comparative transcriptome analysis, we found that secreted proteins involved in responses to nutrient stress are mainly comprised of proteases and glycoside hydrolases. Moreover, 32 secreted proteins undergoing positive selection and 71 duplicated gene pairs encoding secreted proteins are identified. Two duplicated pairs encoding secreted glycosyl hydrolases (GH30), which may be related to fungal endophytic process and lost in many insect-pathogenic fungi but exist in nematophagous fungi, are putatively acquired from bacteria by horizontal gene transfer. The results help understanding genetic origins and evolution of parasitism-related genes.
... It was clear that the isolates were very close to P. chlamydosporia varieties but were not the same. Finally, they carried out a 'molecular host preference' test by amplifying a fragment of the vcp1 gene as described by Morton et al. (2003b). These molecular-based results showed a root-knot 'host preference', although one of the isolates tested, the Pcp21 isolate, showed a putative host preference for both rootknot and cyst nematodes. ...
... chlamydosporia. The amino acid sequence of the VCP1 enzyme shows polymorphism which has been linked to the capacity to infect root-knot or cyst nematodes eggs (Morton et al. 2003b;Siddiqui et al. 2009). The amino acid VCP1 polymorphisms are located in protein sequence positions number 171 and 208. ...
... They used two sets of primers according to host preference or biotype (i.e., root-knot or cyst nematode). The VCP1 polymorphism found by Morton et al. (2003b) was used to design the primers. They tested ten isolates on eggs of root-knot and cyst nematodes. ...
Molecular tools have been increasingly used in identification and taxonomy studies of Pochonia (Clavicipitaceae). In recent years there have been important advances in Clavicipitaceae taxonomy, including resolving the differences between Verticillium and Pochonia through ITS sequences molecular analysis. Current molecular identification of Pochonia spp. relies on different sets of molecular markers to differentiate species, varieties, and host-preference biotypes. The diversity of Pochonia spp. populations has been studied through several genomic fingerprinting techniques, such as RAPD, SCAR, ERIC and REP. Quantification of the fungus in soil and roots can be made using quantitative PCR in combination with other classic microbiological techniques. It is well known that the genus is complex and the current classification is mainly based on the phylogenetic analysis of several genes such as β-tubulin, ITS, nrSSU, nrLSU, rpb1, rpb2 and EF1-α. Molecular advances and recent genome sequencing of Pochonia chlamydosporia has opened a new era in the study of this important fungus, thus broadening the possibilities for studying the molecular mechanisms of differentiation, pathogenesis and diagnostics within Pochonia spp.
... However, the construction of nematode-trapping nets by nematophagous fungi shows that nematophagy is an important trophic state (Jaffe 1992;Jaffe et al. 1992). The egg parasitic fungus P. chamydosporia grows much better on nematode-infested roots than on healthy roots or in the soil (Kerry 2000), and shows a genetic variability closely related to the host from which they were isolated (Morton et al. 2003). These observations suggest that nematodes may be more important to these fungi than being simply an eventual source of nutrients. ...
... These observations suggest that nematodes may be more important to these fungi than being simply an eventual source of nutrients. In the case of obligate parasites, nematode infection initiates through the ingestion of spores or their attachment to the cuticle (Morton et al. 2003). Some endoparasites produce zoospores that are attracted to the nematodes before adhesion and encystment on the cuticle surface. ...
... This group of proteases has a wide spectrum of peptidic substrates, but with preferences for specific substrates. Furthermore, Morton et al. (2003) ob- BAD44716, CAA32820, Q68GV19, CAA63841, AF516146, and AAW21809 correspond to the following organisms and proteases, respectively: Arthrobotrys conoides (cuticledegrading protease), Dactylella avrietas (cuticle-degrading protease), Cordyceps chlamydosporia (VCP1), Paecilomyces lilacinus (pSP-3), Dactylaria parvispora (cuticledegrading protease), Monacrosporium megalosporium (cuticle-degrading protease), Tritirachium album (cuticledegrading protease), Lecanicillium psaliotae (Ver12), Arthrobotrys oligospora (PII), Arthrobotrys oligospora (Aoz1), and Monacrosporium microscaphoides (Mlx) served strong variations at the sequence level and for substrate utilization in isolates from P. chlamydosporia in different nematode hosts. ...
... Thus, fungal penetration is the result of a physic pressure and specific hydrolytic activity of some enzymes such as proteases and chitinases (9). For these reasons, the study of extracellular enzymes and other metabolites excreted by the fungus is an attractive issue for many researchers (10,11,12) due to their possible role as virulence factors or as a criterium for strain selection. A serine protease similar to subtilisin was purified and partially characterized from P. chlamydosporia var. ...
... The VCP1 variability among isolates of P. chlamydosporia can be due to differences in the subtilisins and they can explain the different niches occupied by this facultative parasite in the soil, its host range as well as the virulence factor (11,13). Recently, these differences were shown with the sequencing of the VCP1-encoding gene (12). Up to date, all the studies have been carried out with isolates from the chlamydosporia variety, using the strain 10 from Rothamsted collection as a reference. ...
... min -1 . mL -1 (12). Three replications were done for all determination. ...
The mode of action of Pochonia as a biological control agent is the penetration by means of the hyphae into the target nematode eggs. The egg shell of nematodes has an outer vitelline membrane composed mainly by proteins. Thus, hyphal penetration is the result of a physic pressure and the specific hydrolytic activity of some enzymes such as proteases and chitinases. The most important protease studied in Pochonia chlamydosporia var. chlamydosporia is VCP1. The aim of this work was to detect and characterize the protease VCP1 in a strain of P. chlamydosporia var. catenulata, a potential biological control agent for root-knot nematodes. The specific activity of this protease was tested on different culture media supplemented with protein inductors using N-succinyl-Ala-Ala-Phe-p-nitroanilide as substrate. PCR and RFLP analyses with specific primers and probes were also done. Isolates from the chlamydosporia variety were used as positive controls. The medium supplemented with chitin was the only one capable of inducing some VCP1 activity but in a low level. The VCP1-encoding gene was not detected in strain IMI SD 187 by RFLP even when the stringency conditions were reduced to the minimum possible. The low level of enzymatic activity detected by biochemical techniques does not support the presence of protease VCP1 in this strain. The low enzymatic activity reached points out to the existence of a VCP1 isoform or another different protease. These hypotheses should be proved widening the range of catenulata variety strains and taking the chlamydosporia variety (strain 10) as a reference in a comparative study with conclusive results. The results of this enzymatic characterization related to the egg infection process of host nematodes are the first achieved in a catenulata variety strain in the world.
... ERIC-PCR generated data using in phylogenetic analysis illustrated that the different isolates of the fungus were related to its host from which the isolate had been obtained [330]. Comparison of the similarity of amino acid sequences between proteases from different nematophagous fungi showed a high level of conservation, with only minor insertions and deletions [331]. ...
... Minor variation in amino acid sequence may influence substrate utilization and host preference [51] that has been documented in VCP1 proteases from different isolates of P. chlamydosporia. Substitution of an alanine by a glycine in the S3 substrate-binding region of VCP1 confers enzymatic activity against eggshells of Meloidogyne [330]. ...
... ERIC-PCR generated data using in phylogenetic analysis illustrated that the different isolates of the fungus were related to its host from which the isolate had been obtained [330]. Comparison of the similarity of amino acid sequences between proteases from different nematophagous fungi showed a high level of conservation, with only minor insertions and deletions [331]. ...
... Minor variation in amino acid sequence may influence substrate utilization and host preference [51] that has been documented in VCP1 proteases from different isolates of P. chlamydosporia. Substitution of an alanine by a glycine in the S3 substrate-binding region of VCP1 confers enzymatic activity against eggshells of Meloidogyne [330]. ...
Environmental and health concerns over the use of chemical pesticides have increased the need for alternative measures in the control of plant-parasitic nematodes. Biological control is considered ecologically friendly and a possible alternative in pest and disease management. Several organisms are known to be antagonistic against plant parasitic nematodes. Fungal biological control is an exciting and rapidly developing research area and there is growing attention in the exploitation of fungi for the control of nematodes. In this chapter, important nematode-parasitic and antagonistic fungi are divided into nematophagous and endophytic fungi, and their recent taxonomy, distribution, ecology, biology and their mode of action are discussed.
... The eggs of F. hepatica as they mature, gain some rigidity and endurance, conferring greater protection to the embryo (Toner et al., 2011). In this sense, the rupture of the tegument of the eggs verified in the present study is favored through the action of enzymes present in the fungal extract, among which are the most prominent chitinase VCP1 and carboxypeptidases (SCP1) as reported by Morton et al. (2003). According to Lysek (1976), a species of fungus is considered ovicidal when it has the ability to compromise the process of embryogenesis of helminth. ...
... The prospecting of free proteins originating from the lyophilized fungus was determined in a polyacrylamide gel membrane, where it revealed the presence of proteases of 33 kDa size, corroborating the findings of Segers et al. (1994). This protein is compatible with VCP1, an alkaline serine-protease, secreted by P. chlamydosporia that confers ovicidal potential to the fungus on helminths eggs and gastropod molluscs (Morton et al., 2003;Braga et al., 2010;Duarte et al., 2015;Castro et al., 2019). ...
Hepatic fasciolosis has been implicated as one of the most important parasitic diseases affecting the sanity of cattle and sheep, reflecting in significant economic losses besides being an important anthropozoonotic disease. The eggs of Fasciola hepatica are eliminated with the feces of these hosts, and under the influence of extrinsic and intrinsic factors may remain in the environment for months until they find favorable conditions for embryogenesis. The objective of the present study was to evaluate the viability of Fasciola hepatica eggs exposed for 60 minutes at different concentrations of the enzymatic extract of Pochonia chlamydosporia (Pc-10). Subsequently, they were sedimented and placed on 24-well plates containing the extract of P. chlamydosporia (Pc-10) at concentrations of 500 μL (10%), 400 μL (8%), 300 μL (5%), 200 μL (2%), 100 μL (1%) and distilled water (control group). The experiment was carried out in triplicate, using a total of 900 eggs. After the experimental exposure to the enzyme extract of the fungus (Pc-10), 98% of the eggs exposed to the enzymatic solution demonstrated significant ultrastructural alterations in their respective teguments, when observed in scanning electron microscopy and transmission. The ultrastructure showed a collapse of the internal walls of the egg, interfering in the opening of the operculum. The use of the enzymatic extract of P. chlamydosporia (Pc-10) compromised both the external tegument, breaking it and pasting it, as well as vital structures of the embryonic activity of the F. hepatica eggs.
... Comparison of the similarity of amino acid sequences between proteases from different nematophagous fungi showed a high level of conservation, with only minor insertions and deletions . Minor variation in amino acid sequence may influence substrate utilization and host preference (Segers et al. 1995) that has been documented in VCP1 proteases from different isolates of P. chlamydo-sporia (Morton et al. 2003a, b). Substitution of an alanine by a glycine in the S3 substrate-binding region of VCP1 confers enzymatic activity against eggshells of Meloidogyne (Morton et al. 2003a). ...
... Minor variation in amino acid sequence may influence substrate utilization and host preference (Segers et al. 1995) that has been documented in VCP1 proteases from different isolates of P. chlamydo-sporia (Morton et al. 2003a, b). Substitution of an alanine by a glycine in the S3 substrate-binding region of VCP1 confers enzymatic activity against eggshells of Meloidogyne (Morton et al. 2003a). ...
Plant-parasitic nematodes cause severe damage to world agriculture each year. Environmental and health concerns over the use of chemical pesticides has increased the need for alternative measures to control plant-parasitic nematodes. Nematophagous fungi, a natural enemy of nematodes, have received most attention in biological control of plant-parasitic nematodes. This is due to their specific ability to capture and kill nematodes. Nematophagous fungi are divided into four groups according to their mode of action against nematodes, and several fungi such as Pochonia chlamydosporia and Paecilomyces lilacinus have been developed as commercial biological nematicides. In this chapter, important nematode parasitic and antagonistic fungi, and their taxonomy, biology and their mode of action are discussed. Progress in the study of highly virulent fungal strains for nematode biological control, and application of nematode-antagonistic agents are also discussed.
... Proteaza ta usuwa zewnêtrzn¹ witelinow¹ warstwê z jaj nicieni eksponuj¹c warstwê chitynow¹. Proteaza VCP1 wystêpuje w dwóch izoformach wyizolowanych z dwóch grup nicieni: tworz¹cych cysty oraz guzaków korzeniowych (49). Izoformy te ró¿ni¹ siê pomiêdzy sob¹ dwoma aminokwasami w pozycjach 65 (Glu lub Gln) oraz 99 (Gly lub Ala) (54). ...
... Ró¿nica w wielkooeci reszty aminokwasowej mo¿e w tym przypadku znacz¹co wp³yn¹ae na aktywnooeae enzymu. VCP1 w przeciwieñstwie do proteaz uzyskanych z M. anisopliae oraz A. oligospora nie posiada potencjalnych miejsc N-glikozylacji (49). ...
Microorganisms in biological control of plant-parasitic nematodes S u m m a r y Nematodes are unsegmented roundworms that numerously and success-fully adapted to all regions and environments on earth. The last ones were usu-ally classified into feeding types: free-living, predaceous, and parasitic – in-cluding plant-parasitic. They are of great significance in terms of damage they cause. Plant-parasitic nematodes have been reported to be responsible for the losses amounting to over $100 billion throughout the world. Because of the big difficulties in their eradication some of them are considered as quarantine spe-cies. The plant-parasitic nematodes are controlled using chemical methods – mainly chemical nematicides. However, because of many drawbacks including health and environmental concerns, other control methods are considered. One of them is biological control and application of antagonistic microorganisms to decrease densities of nematodes populations. Microbial antagonists parasitiz-ing various developmental stages of their hosts may affect nematodes by secre-tion of antibiotics, toxins and other secondary metabolites. The most important virulence factors are extracellular enzymes that participate in destroying the nematodes' cuticle or the egg-shell or in further phases of infection. This publication presents the examples of microorganisms investigated in terms of biological control, those that are already available commercially as well as some mechanisms involved in nematode-microbes interactions.
... As a facultative parasite of eggs of plant-parasitic nematodes, the fungus can exhibit different grades of virulence which vary depending on isolate and nematode host. The fungus can be isolated from roots, soil and eggs, and can be cultivated in both liquid and solid media (De Leij & Kerry, 1991;Kerry & Jaffe, 1997;Mauchline, Kerry, & Hirsch, 2002;Morton, Hirsch, Peberdy, & Kerry, 2003). For these reasons, P. chlamydosporia is considered to have great potential in the biological control of root-knot and cyst nematodes (Kerry & Bourne, 2002). ...
... P. chlamydosporia produces three enzymes (protease VCP1, chitinase and lipase) that degrade the eggshell layers of Meloidogyne eggs, but the amount of each enzyme produced varies between isolates. Some authors have suggested that differences in rates of parasitism of eggs amongst isolates originating from soil relate to differences in the diversity of the phytoparasitic nematode communities present in the soil at the time of isolation, and even host preferences within the same isolate (Manzanilla-López, Atkins, Clark, Kerry, & Hirsch, 2009;Morton et al., 2003;Peteira et al., 2009;Siddiqui et al., 2009), as shown molecularly in the present study. The isolates studied here (Table 1) came from soils infested with a variety of phytoparasitic nematodes (Heterodera spp., M. arenaria, M. hapla, M. incognita and N. aberrans) making it reasonable to assume that there would be a diversity in parasitism rates and host preferences of the isolates depending on which nematode species they were Ma. ...
New Mexican isolates of the nematophagous fungus Pochonia chlamydosporia were obtained from nematode infested fields in the vegetable growing area of Tepeaca Valley, Puebla State, Mexico. Based on macro and microscopic morphology, seven ‘putative’ P. chlamydosporia isolates were selected and the DNA extracted for polymerase chain reaction (PCR). Three new isolates of P. chlamydosporia were identified: Pcp2, Pcp21 and Pcp31. The amplification reaction of the internal transcribed spacer (ITS) region revealed a 650 bp amplicon which was used in a maximum likelihood phylogenetic inference analysis. Three groups were recovered in the tree topology, supported by a > 90% bootstrap value. Nucleotide identity values were > 83.6% between the test sequences and the reference sequence. In addition, using specific primers for two existing varieties of P. chlamydosporia, restriction fragment length polymorphism on the ITS products in conjunction with the phylogenetic inferences and the molecular test for detection of P. chlamydosporia vcp1 gene, it was found that all three isolates belong to a new variety which we have named P. chlamydosporia var. mexicana. We compared the chlamydospore production rate, rhizosphere colonisation and egg parasitism percentages of the three native isolates in Meloidogyne spp. with a reference isolate (Pc10). Native isolates produced > 1×106 chlamydospores/50 g of substrate (of which more than 80% were viable), colonised > 80% of the rhizosphere, and parasitised > 60% of Meloidogyne incognita and Meloidogyne arenaria eggs. Meloidogyne hapla egg parasitism was Meloidogyne spp. to be tested further in greenhouse and field tests.
... Similar to Metarhizium, P. chlamydosporia also showed the ability to colonize roots of some plants and to improve plant growth (Bordallo et al., 2002;Maciá-Vicente et al., 2009b). However, P. chlamydosporia differs in that it is a nematophagous fungus and previous research has compared the endophytic and pathogenic capabilities of Metarhizium and P. chlamydosporia (Aranda-Martinez et al., 2017;Moonjely and Bidochka, 2019;Morton et al., 2003). ...
... Resistance to all classes of broad-spectrum anthelmintics has already been reported [2]. As time has passed problems of multi-resistance to more than one class have occurred and the multi-resistant nematode has become a major threat to the whole small ruminant industry [3]. ...
Background
Several species of nematophagous fungi exist in nature that can capture and kill nematodes as natural predators of soil-dwelling worms. These are important in agriculture and animal husbandry as biological control agents. The diversity of nematophagous fungi found from soil had not been studied in Ethiopia.
Objective
This study aimed to isolate Nematophagous Fungi from Soil Samples Collected From three Different Agro-Ecologies of Ethiopia.
Methods
Cross-sectional study was conducted and samples were collected from three different agro-climatic zones of Ethiopia; Debre-Berhan (highland), Bishoftu (mid-altitude), and Awash (lowland). Twenty-seven soil samples were randomly taken from each of the three different agro-ecological climates (9 from each agro-ecological climatic zone). For each study site, samples were collected from the soil of decomposed animal feces/dung, agricultural/farmlands, and forest lands in triplicates.
Results
The present study disclosed that nematophagous fungi were widespread from the study area. A total of 33 species of nematophagous fungi belonging to four genera, Arthrobotryes, Paecilomyces, Monacrosporium, and Harposporium were identified. Arthrobotrys were the most commonly isolated genera followed by Paecilomyces. The six identified species were Arthrobotrys oligospora, Paecilomyces lilacinus, Arthrobotryes dactyloides, Monacosporum eudermatum, Harposporium helicoides, and Monacosporum cionopagum.
Conclusion
This study indicated that Arthrobothryes oligospora was the most common species in Bishoftu and Awash whereas. In Debre-Berhan, Paecilomyces lilacinus was the most prevalent species. Monacosporum cionapagum was not isolated from dung soil and agricultural soil whereas Harposporium helicoides and Arthrobothryes dactyloides were not found from dung and forest soil respectively.
... formed a different group to those infested with Meloidogyne spp., thereby suggesting that genetic variation and adaptation relate to Pathogens 2022, 11, 619 7 of 12 the host. The capacity to infect RKN or CN eggs has been linked to amino acid sequence polymorphism of the VCP1 serine protease [32]. However, for some isolates, the vcp1 gene polymorphism of isolates from CN or RKN do not always match the original nematode host or biotype reported [7]. ...
The endophytic and nematophagous fungus Pochonia chlamydosporia is an efficient biological control agent of plant-parasitic nematodes. Isolates of the fungus can be allocated to a biotype group according to the nematode host, but it is unknown if genetic interchange can occur between different biotypes, which may affect their parasitic performance. An anastomosis assay was conducted in vitro to assess hyphae vegetative compatibility/incompatibility followed by a PCR-based mating-type assay genotyping of five isolates of P. chlamydosporia var. chlamydoporia of the Meloidogyne sp. (Pc10, Pc190, Pc309), Globodera sp. (Pc280) and Heterodera avenae (Pc60) biotypes, including 16 pairwise isolates combinations in four replicates. Pairwise combinations were tested on glass slides and mycelia were stained to confirm nuclei migration between anastomosing hyphae using fluorescence microscopy. Anastomosis only occurred between mycelium hyphae of the same isolate and biotype. Mating-type PCR-based molecular assays showed that all isolates were heterothallic. The MAT1-1 genotype was found in isolates Pc10, Pc190, Pc280, Pc309, and the MAT1-2 genotype in Pc60. The results showed a vegetative incompatibility among isolates, suggesting the occurrence of such interactions for their respective biotypes. Anastomosis and PCR mating-type results suggest that different fungal biotypes can occur in the same niche but that genetic incompatibility mechanisms, such as mating-type, may limit or impede viable heterokaryosis.
... In addition to promoting efficient control of nematodes, when in their absence, P. chlamydosporia is able to survive in the rhizosphere of many plants and to use organic matter as an additional source of nutrients (Coutinho et al., 2009), which means that is a desirable feature for field application. Furthermore, it is an optional parasite of eggs of gall and cyst nematodes and its infectious process involves the production of enzymes that remove the protein membrane from the egg, exposing the underlying chitin layer (Morton et al., 2003). ...
The rhizosphere is the region that has direct influence from the roots. This is the place where most of the interactions between microorganisms and plants occur. Studies involving the ecology of microbial communities from the rhizosphere became more frequent after the first reports of biological interactions of microorganisms with plants that influence physically and chemically modify the soil surrounding. According to these hypotheses, the rhizosphere mycobiota provides the development of plants through various mechanisms, direct and indirect. Thus, the objective of this review was to explain the aspects that provide characterizing these microorganisms as beneficial to plants in view of their applicability to agro-ecosystems. Therefore, it is stated that rhizospheric fungi have the solubilization of phosphorus (P), assimilating this nutrient for plants, promoting growth through the production or stimulation of the production of growth regulators such as 3-indole acetic acid, and control of phytopathogenic agents such as other filamentous fungi, and also phytonemamatodes. Therefore, it is possible to observe the importance of the constant observance of the action of these microorganisms in terms of their ecological role due to the agro-ecosystem.
... Parasitic ability: The serine protease VCP1 of P. chlamydosporia is involved in the early stages of the infection process (Morton et al., 2003a(Morton et al., , 2003b. Primers for the detection of the vcp1 gene from P. chlamydosporia and a PCR assay for host preference to distinguish between biotypes of the fungus from CN and RKN have been designed. ...
... The protein sequences of Pr1 (St. Leger et al. 1992), VCP1 (Morton et al. 2003) and Ver112 (Yang et al. 2005) were found in the GenBank database, and the signal peptide and pro-peptide sequences were omitted to obtain mature proteins with 281, 281 and 280 amino acids for Pr1, VCP1 and Ver112, respectively (Liu et al. 2007). The results revealed some physiochemical properties, namely, a comparable amino acid residue number (approximately 280), a low molecular weight (approximately 28.6 kDa), a pI value more than 7.7 and inhibition by phenyl methane sulfonyl fluoride (Liu et al. 2007). ...
Entomopathogenic fungi are among the most successful
biocontrol agents for preventing economic loss from insects.
The identification of virulent species or isolates, the development
of formulation technology and the improvement of
efficiency are avenues being pursuing by researchers in
diverse scientific disciplines. A successful entomopathogenic
fungus deploys a combination of mechanical and biochemical
processes to overcome the first defensive barrier in
insects, the integument. A precise understanding of the
mechanisms underlying fungal pathogenicity, particularly
the roles of enzymes such as proteases, is essential in order
to highlight the potential of entomopathogenic fungi and
increase their virulence via genetic modifications. Cuticledegrading
proteases are divided into subtilisin-like (Pr1) and
trypsin-like (Pr2) proteases, which are secreted in the initial
stages of penetration. The biochemical structure contains
the catalytic triad Asp39, His69 and Ser224 in addition to
Ca2þ binding sites. Studies have shown a molecular weight
of almost 19–47 kDa, an optimal pH of 7–12 and an optimal
temperature of 35–45 �C. Different species or isolates of
entomopathogenic fungi exhibit differences in the secretion
and activity of cuticle-degrading proteases, which may indicate
their virulence capacity. Genetic engineering techniques
have been developed to create isolates with protease
overexpression. Such isolates have significantly higher virulence
against the host because they not only ensure fungal
penetration but also exhibit direct toxicity to insects.
... Host preference in isolates from cyst and root-knot nematodes was reported by Mauchline et al. (2004) and Manzanilla-Lopez et al. (2011). The association has been related to specific amino acid polymorphisms in a fungal serine protease, VCP1, involved in the infection of nematode eggs (Morton et al. 2003). The enzyme degrades the proteinaceous vitelline membrane, the outer layer of the nematode eggshell, which is qualitatively different in root-knot and cyst nematodes (Morton et al. 2004). ...
The biocontrol potential of four Pochonia chlamydosporia var. chlamydosporia isolates on the root-knot nematode, Meloidogyne javanica was investigated on pistachio plants in a greenhouse experiment. Isolates were applied at 10,000, 5000 and 1000 chlamydospores per gram of soil; the latter two densities were being used for strain Pcc60 only and designated as PccB and PccA, respectively. Plants were inoculated with a suspension containing 3000 eggs of M. javanica. Nematode reproduction was reduced to 57% by Pcc20 and 36% by Pcc10 and Pcc60C after 4 months. Pcc20 was the most promising isolate as the final egg population of nematodes was reduced by nearly 61 and 36% by Pcc10 and Pcc60C, respectively. All strains infected eggs on the roots to varying degrees. Pcc20 was the most effective colonizer of all the isolates, infecting 37% of the eggs, while Pcc10 and Pcc60C as the second best colonizers caused an average infection rate of 17.5%. The potential of the fungus was reduced at lower densities of spore inoculum. Semi-selective medium was used to re-isolate the fungi from soil and rhizosphere but was not able to detect Pcc60A at 1000 cfu/g soil, despite the fact that 3% of the eggs were parasitized. The efficiency of the different isolates in controlling nematode reproduction parameters and their relationships in terms of survival and abundance is discussed further.
... 30 Importantly, intriguing evidence emerged suggesting that subtilisin-like serine proteases isolated from nematode-trapping fungi regulate important processes of penetration, degradation, and digestion of nematode cuticles. [34][35][36][37][38][39] Several kinds of serine protease including PII and Aoz1 from Arthrobotrys oligospora, 40 pSP-3 from Paecilomyces lilacinus 41 and VCP1 from Paecilomyces chlamydosporia 42 have been isolated, purified and cloned. These enzymes disrupt the interrupt the physiological integrity of the nematode cuticle that facilitates penetration and colonization. ...
The crop loss incurred by the infestation of wide range of nematodes is valued in billions of dollars and has posed a serious threat to the global food security. The nematode affects economically important crops all over the world and incurs a significant decline in the crop
yield. Management of nematodes is given consideration and different methods have been developed. Intriguing evidence suggest that the biological method to control nematode infestation is the best and viable approach. Control of nematodes by the use of biocontrol
agents offers a promising substitute to the use of chemical agents and chemical associated harmful effects. The microbes such as fungi and bacteria have the ability to reduce the nematode population and have played a vital role in decreasing the crop yield loss caused by
nematodes. The development and subsequent implementation of biocontrol agents is very unpredictable and quite hectic on a large-scale. The implementation of an ideal biocontrol agent requires an elaborated understanding of the mechanisms of infectivity of the antagonist against nematode populations, and meticulous exploration of the interactions among biocontrol isolates, nematode, soil micro biota, plant microenvironment and ecological implication must be developed.
... For VCP1 puriication P. chlamydosporia was grown in soy peptone medium, and VCP1 was puriied from culture ltrates by isoelectric focusing. Both, VCP1 and P32 serine proteases were cloned ( Morton et al. 2003;Larriba et al. 2012). A new, ca 60 kDa, serine carboxy- peptidase from Pochonia spp. ...
Fungal proteomics research is growing as a result of the large number of fungal sequenced genomes of well annotated proteins available today. The proteome of any organism is dynamic as proteins differ depending on environmental conditions, unlike genomes which are practically constant for all the cells of an organism. In this chapter we have reviewed the ‘state-of-the-art’ of fungal proteomics, including sample preparation, protein separation and identification. We have given examples of proteomics of entomopathogenic and nematophagous fungi. We have also focused our attention on the proteomic study of Pochonia chlamydosporia carried out to date. In this study, the fungus was grown in chitin or chitosan as the main carbon and nitrogen nutrient sources, and the secretome of the fungus in both conditions analyzed. Proteins were concentrated using TCA/acetone. Two-dimensional, sodium dodecyl sulphate polyacrylamide gel electrophoresis and differential gel electrophoresis separated proteins for size and isolectric point. Some of the proteins overexpressed with chitosan that were identified using MALDI/TOF-TOF and LC-MS, were related with carbohydrate or protein degradation. The recently available complete Pochonia chlamydosporia genome sequence could help with protein identification of fungal secretomes under various conditions.
... Fungal isolates are often referred to as biotypes according to the nematode hosts from which they were originally isolated: root-knot nematode (RKN biotype), potato cyst nematode (PCN biotype), cereal cyst (CN biotype). Biotypes can show a host preference, a feature that is related to differences in egg parasitism associated with specific amino acid polymorphisms in the fungal serine protease VCP1, at least for root-knot and cyst nematodes (Morton et al. 2003a, b). Serine proteases enzymes are putative determinants of pathogenicity or virulence (Butt et al. 1998). ...
The nematophagous fungus Pochonia chlamydosporia (Clavicipitaceae) is a facultative parasite of major plant-parasitic nematodes pests such as cyst (Globodera spp., Heterodera spp.), root-knot (Meloidogyne spp.), false root-knot (Nacobbus spp.) and reniform (Rotylenchulus reniformis) nematodes. The potential of P. chlamydosporia as a biological control agent and biopesticide has been the subject of numerous studies aimed at understanding the micro-ecological conditions that allow the fungus to thrive in the soil and rhizosphere environments. Pochonia survives in soil in the absence of plant and nematode hosts and can also behave as an endophyte. Research evidence points to a physiological ‘switch’ from the saprophytic to the parasitic stage that is triggered by nutrition. The basic biology of the fungus and sedentary endoparasitic plant nematodes is reviewed to provide insights into the fungus multitrophic behaviour, as well as its importance as a biocontrol agent within an integrated pest management approach.
... We have found that P. chlamydosporia generates the best ethanol yield. This fungus presents a worldwide distribution and genetic variations, enzyme production and plant promoting capabilities among P. chlamydosporia isolates have been related to their geographical origin (Morton et al., 2003;Esteves et al., 2009;Zavala-Gonzalez et al., 2015). P. chlamydosporia strain selection would perhaps enable an increase in ethanol yield above that achieved in the current study. ...
... Upon contact with nematode eggs, P. chlamydosporia colonizes the surface of the egg forming numerous appressoria and penetration pegs (Escudero and Lopez-Llorca 2012 ;Segers et al. 1996 ). An alkaline serine protease designated VCP1 is involved in infection of nematode eggs by P. chlamydospora (Morton et al. 2003a ). Polymorphisms w ithin the gene encoding this enzyme are related to host preference for either cyst or root-knot nematodes among fungal isolates. ...
Plant-parasitic nematodes and microfungi inhabit many of the same ecological habitats and interact in almost every conceivable way. Nematodes can feed on fungi, and conversely fungi can use nematodes as a food source. Fungi have been widely studied as biological controls of plant-parasitic nematodes. Fungi can attract or repel nematodes, and nematodes and fungi can interact to either directly or indirectly increase or even decrease plant disease. Nematodes can also feed on fungi and act as biological controls of plant disease. Plant-parasitic nematodes likely obtained the cell wall-degrading enzymes necessary to successfully feed on plants from fungi through horizontal gene transfer. Finally, plant-parasitic nematodes can interact with fungal pathogens or even nonpathogenic or weakly pathogenic fungi to increase plant disease.
... P. chlamydosporia secretes the serine protease VCP1 and a newly found serine carboxypeptidase (SCP1), when endophytically colonizing barley roots . VCP1 and SCP1 have been also cloned, characterized and inmunolocalized in M. javanica eggs infected by the fungus (Morton et al., 2003;Larriba et al., 2012;Escudero et al., 2016). However, the mechanisms involved in the interaction between the fungus, the nematode and the plant root should be better understood in order to improve the performance of P. chlamydosporia as a biocontrol agent. ...
Pochonia chlamydosporia is a soil fungus with a multitrophic lifestyle combining endophytic and saprophytic behaviors, in addition to a nematophagous activity directed against eggs of root-knot and other plant parasitic nematodes. The carbohydrate-active enzymes encoded by the genome of P. chlamydosporia suggest that the endophytic and saprophytic lifestyles make use of a plant cell wall polysaccharide degradation machinery that can target cellulose, xylan and, to a lesser extent, pectin. This enzymatic machinery is completed by a chitin breakdown system that involves not only chitinases, but also chitin deacetylases and a large number of chitosanases. P. chlamydosporia can degrade and grow on chitin and is particularly efficient on chitosan. The relevance of chitosan breakdown during nematode egg infection is supported by the immunolocalization of chitosan in Meloidogyne javanica eggs infected by P. chlamydosporia and by the fact that the fungus expresses chitosanase and chitin deacetylase genes during egg infection. This suggests that these enzymes are important for the nematophagous activity of the fungus and they are targets for improving the capabilities of P. chlamydosporia as a biocontrol agent in agriculture.
... In 1988, an extracellular protease was detected in Verticillium suchlasporium, a nematophagous fungus endoparasite of nematode cysts, being the first serine protease purified (P32) (Lopez-Llorca, 1990). Other serine proteases from nematophagous fungi were purified and cloned, including PII and Aozl of Arthrobotrys oligospora (Tunlid and Jasson, 1991;Tunlid et al., 1994;Ahman et al., 1996;Zhao et al., 2004), pSP-3 of Paecilomyces lilacinus (Bonants et al., 1995), and VCP1 of Pochonia chlamydosporia (Segers et al., 1994;Morton et al., 2003). ...
... (particularly T. longibrachiatum and T. harzianum), Paecilomyces lilacinus, and Dactyella ovoparasitica. Some of them, like P. chamydosporia, grow much better on nematode infested roots than on healthy roots or in the soil (Kerry 2000) and show a genetic variability closely related to the host from which they were isolated (Morton et al. 2003), suggesting that the nematodes may be more important to these fungi, than just an eventual source of nutrients. ...
Nematophagous fungi represent an ancient and diverse group of fungi that use refined mycelial structures or their conidia to capture. They are classified on the basis of their mechanism of interaction with the animal as endoparasites, trap-forming fungi, and opportunists. They have received high attention as biological control agents against nematodes, but also as model organisms for “carnivorous” eukaryotic microorganisms. Here, we review the current state of knowledge of their biology and molecular physiology and particularly highlight the recent genomic insights into the virulence factors of nematophagous fungi.
... Further work is necessary to develop a primer and probes set that is specific to this isolate. At present, sequence data are limited with only ITS and serine protease genes [28] having been sequenced and would not provide regions suitable for selective primers to be developed. ...
The fungus Pochonia chlamydosporia var. catenulata has a potential as a biological control agent against root-knot nematodes. If the fungus was applied on rice established in the soil, it would provide a cheaper form of application of the fungus for local farmers. Two types of fungal inocula were tested: a) the fungus added on colonised rice grain and, b) the purified chlamydospore powder. Each was added to plots infested with root-knot nematodes and the plots planted with two consecutive tomato crops. The population of P. chlamydosporia var. catenulata was monitored using real-time PCR and plating soil onto a selective medium for the fungus. The fungal inoculum added to rice established and proliferated more successfully than it did when it was added as a chlamydospore powder. However, there was no significant difference between the proportion of eggs infected after both fungal treatments at the first harvest, or in fungal densities measured as CFU or DNA levels by the time of the planting of the second tomato crop. Baiting the soil with nematode egg masses at different times after application demonstrated that the fungus applied in both forms infected significantly more eggs than soil from the untreated control plots. Pochonia chlamydosporia var. catenulata significantly reduced pest nematode populations and could be successfully used as a soil amendment.
... (Guerrero et al. 2010) that, along with chitinases, are considered important for Pc 427 parasitism of nematode eggs (Huang et al. 2004; Lopez-Llorca et al. 2002; Mi et al. 428 2010; Morton et al. 2003; Segers et al. 1994; Tikhonov et al. 2002). Pochonia spp. ...
Pochonia chlamydosporia (Pc), a nematophagous fungus and root endophyte, uses appressoria and extracellular enzymes, principally proteases, to infect the eggs of plant parasitic nematodes (PPN). Unlike other fungi, Pc is resistant to chitosan, a deacetylated form of chitin, used in agriculture as a biopesticide to control plant pathogens. In the present work, we show that chitosan increases the incidence and severity of Meloidogyne javanica egg parasitism by P. chlamydosporia. Using antibodies specific to the Pc enzymes VCP1 (a subtilisin), and SCP1 (a serine carboxypeptidase), we demonstrate chitosan elicitation of the fungal proteases during the parasitic process. Chitosan increases VCP1 immuno-labelling in the cell wall of Pc conidia, hyphal tips of germinating spores, and in appressoria on infected M. javanica eggs. These results support the role of proteases in egg parasitism by the fungus and their activation by chitosan. Phylogenetic analysis of the Pc genome reveals a large diversity of subtilisins (S8) and serine carboxypeptidases (S10). The VCP1 group in the S8 tree shows evidence of gene duplication indicating recent adaptations to nutrient sources. Our results demonstrate that chitosan enhances Pc infectivity of nematode eggs through increased proteolytic activities and appressoria formation and might be used to improve the efficacy of M. javanica biocontrol.
... However, one isolate from the same or different nematode biotypes (i.e., from the original nematode host) can dominate over other isolates when sharing the same habitat (Manzanilla-López et al., 2009a, b). Fungal biotypes can also show a host preference at the infraspecific level, a feature that is related to differences in egg parasitism associated with specific amino acid polymorphisms in the fungal serine protease VCP1 (Morton et al., 2003a). Plants also differ in their host status, not only for the fungus, but also for the nematode. ...
The nematophagous fungus Pochonia chlamydosporia (Clavicipitaceae) can parasitize eggs of nematodes including cyst (Globodera spp., Heterodera spp.), root-knot (Meloidogyne spp.), false-root knot (Nacobbus spp.) and reniform (Rotylenchulus reniformis) nematodes. Its potential as a biological control agent has been the subject of numerous studies aimed at understanding the micro-ecological conditions, including the tritrophic (e.g., plant, fungus, nematode) and host-parasite relationships that allow the fungus to thrive in the soil and rhizosphere environment. In order to effectively exploit the fungus in regulating plant endo-parasitic nematodes, a careful selection of fungal isolates (biotypes) appropriate for both host plant and nematode is essential. Pochonia survives in soil in the absence of a nematode host and, although it behaves as a saprophyte, research evidence points to a physiological 'switch' from the saprophytic to the parasitic stage that is triggered by nutrition. Fungal chlamydospore-based formulations for application to soil as an inoculum have been shown to be viable commercial formulations. The basic biology and physiology of the fungus is herein reviewed to provide new insights into the host-parasite interaction and commercial production methodology.
... In 1988, an extracellular protease was detected in Verticillium suchlasporium, a nematophagous fungus endoparasite of nematode cysts, being the first serine protease purified (P32) (Lopez-Llorca, 1990). Other serine proteases from nematophagous fungi were purified and cloned, including PII and Aozl of Arthrobotrys oligospora (Tunlid and Jasson, 1991;Tunlid et al., 1994;Ahman et al., 1996;Zhao et al., 2004), pSP-3 of Paecilomyces lilacinus (Bonants et al., 1995), and VCP1 of Pochonia chlamydosporia (Segers et al., 1994;Morton et al., 2003). ...
... showing the greatest versatility for different enzymatic substrates. Other studies involving strains of the genus Pochonia have shown that this genus is also a producer of extracellular proteases in the presence of gelatine [79][80][81][82]. ...
Entomopathogenic fungi can regulate insect populations. They have extracellular enzymes that degrade cuticle components, mainly hydrocarbons, used as an energy source. The increase in insecticidal activity of fungi in a medium supplemented with cuticular hydrocarbons was assayed and the hydrolytic enzyme profiles of two strains of Purpureocillium lilacinum were evaluated. A spore suspension of P. lilacinum was inoculated in Petri plates with different values (0.99–0.97–0.95) of water activity (Aw) using the substrates gelatin, starch and tween-20. Growth rate on the different substrates and the enzymatic activity index for proteases, amylases and lipases at different incubation times, pH and Aw, was evaluated. Moreover, the insecticidal efficiency of strains grown in media supplemented with n-hexadecane and n-octacosane was analyzed. LT50 was calculated against adults of Tribolium confusum and showed that mortality increased about 15% when the strains grew in amended culture medium. High amylolytic activity was detected, but proteases were the main enzymes produced. Optimal protease production was observed in a range of acid and alkaline pH and lower Aw. The greatest growth rate was obtained in presence of gelatin. Lipase and amylase production was detected in small amounts. Fungal growth in media with hydrocarbon mixtures increased the pathogenicity of the two strains of P. lilacinum, with the strain JQ926223 being more virulent. The information obtained is important for achieving both an increase in insecticidal capacity and an understanding of physiological adaptation of the fungus.
... The lytic activity of the fungal enzymes is influenced by the substrate specificity of the enzymes (Dackman et al., 1989;Esteves et al., 2009;Lýsek et al., 1982;Morton, Hirsch, Perberdy, & Kerry, 2003;Segers et al., 1996). Lýsek (1976) suggested a classification system for fungal ovicidal effect based on lytic activity on the egg shell categorising the effect into three types. ...
Thick-shelled eggs of animal-parasitic ascarid nematodes can survive and remain infective in the environment for years. The present study evaluated a simple in vitro method and evaluation scheme to assess the relative effect of two species of soil microfungi, Pochonia chlamydosporia Biotype 10 and Purpureocillium lilacinum Strain 251 (Ascomycota: Hypocreales), on the development and survival of eggs of faecal origin of three ascarid species, Ascaridia galli (chicken roundworm), Toxocara canis (canine roundworm) and Ascaris suum (pig roundworm). Ascarid eggs were embryonated on water agar with or without a fungus, and the resulting viability of the eggs was evaluated on days 7, 14, 21, 28, 35 and 42 post exposure (pe) by observing eggs in situ. On days 7–42 pe, P. chlamydosporia had reduced the viability of A. galli and T. canis eggs by 64–86% and 26–67%. Corresponding reductions for P. lilacinum Strain 251 were
only 15–29% and 4–28%. In contrast, A. suum eggs were extremely resistant to both fungi (2–4% reduction). The differences in results are likely due to different morphologies and chemistry of the egg shell of the three ascarid species. The current in vitro method and evaluation criteria allow for a simple, repeatable and
non-invasive evaluation of the ovicidal effects of microfungi. This study demonstrates that P. chlamydosporia Biotype 10 may be utilised as a biocontrol agent to reduce A. galli and T. canis egg contamination of the environment.
... Our results are consistent with previous results on the nuclear genome, in which the genome of P. chlamydosporia was found to be most closely related to entomopathogenic fungi Metarhizium spp. [11], as well as on the phylogenetic analyses of the P. chlamydosporia serine proteases [41,42]. Our phylogenetic results also show that fungi attributed to the same genus group together at first, such as the three species of Beauveria (B. ...
Background
The fungus Pochonia chlamydosporia parasitizes nematode eggs and has become one of the most promising biological control agents (BCAs) for plant-parasitic nematodes, which are major agricultural pests that cause tremendous economic losses worldwide. The complete mitochondrial (mt) genome is expected to open new avenues for understanding the phylogenetic relationships and evolution of the invertebrate-pathogenic fungi in Hypocreales.ResultsThe complete mitogenome sequence of P. chlamydosporia is 25,615 bp in size, containing the 14 typical protein-coding genes, two ribosomal RNA genes, an intronic ORF coding for a putative ribosomal protein (rps3) and a set of 23 transfer RNA genes (trn) which recognize codons for all amino acids. Sequence similarity studies and syntenic gene analyses show that 87.02% and 58.72% of P. chlamydosporia mitogenome sequences match 90.50% of Metarhizium anisopliae sequences and 61.33% of Lecanicillium muscarium sequences with 92.38% and 86.04% identities, respectively. A phylogenetic tree inferred from 14 mt proteins in Pezizomycotina fungi supports that P. chlamydosporia is most closely related to the entomopathogenic fungus M. anisopliae. The invertebrate-pathogenic fungi in Hypocreales cluster together and clearly separate from a cluster comprising plant-pathogenic fungi (Fusarium spp.) and Hypocrea jecorina. A comparison of mitogenome sizes shows that the length of the intergenic regions or the intronic regions is the major size contributor in most of mitogenomes in Sordariomycetes. Evolutionary analysis shows that rps3 is under positive selection, leading to the display of unique evolutionary characteristics in Hypocreales. Moreover, the variability of trn distribution has a clear impact on gene order in mitogenomes. Gene rearrangement analysis shows that operation of transposition drives the rearrangement events in Pezizomycotina, and most events involve in trn position changes, but no rearrangement was found in Clavicipitaceae.Conclusions
We present the complete annotated mitogenome sequence of P. chlamydosporia. Based on evolutionary and phylogenetic analyses, we have determined the relationships between the invertebrate-pathogenic fungi in Hypocreales. The invertebrate-pathogenic fungi in Hypocreales referred to in this paper form a monophyletic group sharing a most recent common ancestor. Our rps3 and trn gene order results also establish a foundation for further exploration of the evolutionary trajectory of the fungi in Hypocreales.
... Studies of parasitic fungi revealed that the chemical composition of the host surface is an important inducer for the production of specific hydrolytic enzymes, involved in infection (Tunlid & Jansson, 1991). In the P. chlamydosporia-nematode interaction, most of the knowledge about the fungus molecular responses concerns VCP1, an enzyme that specifically degrades proteins from the outer egg shell layers (Segers et al., 1996;Morton et al., 2003). However, VCP1 release represents a single step in a more complex biochemical cascade of gene induction and activation processes, largely unknown (Lopez-Llorca et al., 2010;Ward et al., 2012). ...
In vitro assays were performed under varying nutritional conditions to investigate the expression of four genes of the fungus Pochonia chlamydosporia, a nematode parasite and a plant growth promoter. The expression levels of a basic leucine zipper (bZIP) transcription factor, a putative phytase, a phospholipase D (PLD) and a monoxygenase of isolate DSM 26985 were, after 8 h incubation in the presence of Meloidogyne incognita eggs, 3, 14, 900 and 67‐fold higher than the fungus alone, respectively. M. incognita uninoculated tomato plants alone induced, at 4 h incubation, a phytase transcription sixfold higher than control. Changes in transcript amounts were not significant when the fungus was in the presence of nematode parasitized plants. Further assays performed at different pHs or in the presence of glucose and NH4+ showed that the bZIP and phytase transcripts reflect early changes in the fungus metabolism. Barley plants treated with P. chlamydosporia isolates IMI 331547 and DSM 26985 showed a pronounced plant growth promotion effect of the former isolate. Phytic acid affected growth of isolates at 1 × 10−4 ppm concentration, however, the colony diameter increased at 10 ppm. Gene expression data showed that P. chlamydosporia metabolism reflects key biochemical signals proceeding from roots and host nematode eggs.
... Hasta el presente, los mecanismos que confieren al hongo especificidad frente al nematodo hospedante no están claros. Diferentes autores encontraron a partir del análisis de diferentes aislamientos por PCR-ERIC, que era posible agrupar los materiales estudiados de acuerdo al hospedante, planteando que esto pudiera indicar que hay elementos en la población de P. chlamydosporia que están asociados con la infección de un género particular de nematodos (10,11,12). Morton et al. (9) encontraron polimorfismo en la composición aminoacídica de la VCP1, al comparar aislamientos provenientes de diferentes hospedantes. ...
The Cuban strain IMI SD 187 of the nematophagous fungus Pochonia chlamydosporia var. catenulata is a potential biological control agent for the root knot nematodes. There are reports about the controlling action of these fungi on cyst nematodes. However, isolate specificity according to the original host is also known. The aim of this work was to study the enzymatic performance induced by Meloidogyne incognita and Globodera pallida eggs. The induction assays were carried out in liquid media. The total protein content and the enzymatic activity levels for proteases, chitinases, lipases and VCP 1 were determined. The time course experiment for enzymatic activity induction was related to the infection process of M. incognita eggs by the strain IMI SD 187. The strain IMI SD 187 of P. chlamydosporia var. catenulata isolated from root knot nematodes is able to infect eggs from cyst nematodes.
Sedentary phytonematodes are economically important pests that should sustainably be managed. Chemical nematicides have continued as the most routine controlling methods but must be substituted with other safer methods because of their adverse impact on environmental pollution. The application of fungal biocontrol agents or their derivatives is a promising method that has attracted the attention of many researchers. Pochonia, Purpureocillium, Trichoderma, and Lecanicillium are the most important egg-parasitic fungi frequently associated with suppressive soils. Parasitic fungi can antagonize phytonematodes mainly by direct parasitism, stimulating plant resistance and promoting its growth. Many egg and female-parasitic fungi have endophytic lifestyles whose interactions with roots may up-regulate salicylic acid-dependent resistance or pathogenesis-related genes. The mentioned characteristics make egg and female parasitic fungi a promising substitute for managing phytonematodes. This chapter reviews current taxonomy, biology, ecology, and mode of action of egg-parasitic fungi and show their significance against phytonematodes. However, more in-depth studies about the factors that enhance the virulence of those fungi and new genetic tools will improve the possibility of their success as a reliable method to produce robust and durable nematode control.
Plant-parasitic nematodes (PPNs) cause severe damage to agricultural crops worldwide. As most chemical nematicides have negative environmental side effects, there is a pressing need for developing efficient biocontrol methods. Nematophagous microbes, the natural enemies of nematodes, are potential biocontrol agents against PPNs. These natural enemies include both bacteria and fungi and they use diverse methods to infect and kill nematodes. For instance, nematode-trapping fungi can sense host signals and produce special trapping devices to capture nematodes, whereas endo-parasitic fungi can kill nematodes by spore adhesion and invasive growth to break the nematode cuticle. By contrast, nematophagous bacteria can secrete virulence factors to kill nematodes. In addition, some bacteria can mobilize nematode-trapping fungi to kill nematodes. In response, nematodes can also sense and defend against the microbial pathogens using strategies such as producing anti-microbial peptides regulated by the innate immunity system. Recent progresses in our understanding of the signal pathways involved in microbe–nematode interactions are providing new insights in developing efficient biological control strategies against PPNs.
This article is part of the theme issue ‘Biotic signalling sheds light on smart pest management'.
Abstract
In the present study influence of pH and temperature on extracellular enzymic activity of protease on
solid media were tested of six species of nematode -trapping fungi (Arthrobotrus anchonia , A.dactyloides ,
A.oligospora , Dactylella brochopaga , Dactyleria pyriformic , Monacrosporium eudermatum) . The
extracellular enzymic activity of protease were varied according to fungal species and the value of pH and
temperature . A.oligospora produced high vegetative growth in all pH values , followed by M.eudermatum ,
whereas D.pyriformic showed a low vegetative growth followed by A.anchonia . Nevertheless, all tested
fungi showed a high vegetative growth at pH = 7.5 , followed by value 7 , whereas it's growth was weak at
pH = 8.5 , no vegetative growth showed at pH = 6 .
A.oligospora showed a high enzymic activity followed by M.eudermatum , a low enzymic activity was
appeared in D.pyriformic . A best enzymic activity in the tested fungi produced at pH = 7.5 , followed by pH
= 7 , while all tested fungi were never showed enzymic activity in pH = 6 and 8.5 .
This Study showed that the M.eudermatum produced a highest growth rate in all tested temperature degrees ,
followed by A.oligospora , whereas D.pyriformic showed lowest growth rate , the vegetative growth in other
tested fungi were varied
However, the optimum temperature for vegetative growth of all tested fungi was 25 C° , followed by 30 C° ,
while the lowest vegetative growth was appeared at 40C° followed by 15C° . The high activity of protease
showed in A.oligospora , followed by M.eudermatum , the low activity revealed in D.brochopaga, in tested
temperature degrees . the 30C° showed a high enzymic activity . followed by 25C° , on the contrary , no
enzymic activity appeared in all tested fungi in both 15C° and 40C° .
Keywords: Nematode -trapping fungi , protease , pH , temperature , extracellular enzymic activity.
Pochonia parasitizes the eggs and females of plant-parasitic nematodes (PPN). The fungus-nematode interaction involves a complex series of events which can be affected by the environment. Thus, an understanding of the bionomics of this interaction is essential in order to improve the efficiency of the biological management of PPN. In this chapter, we provide an overview of the process of infection of nematodes by Pochonia and the role of the environment on the fungus-nematode interaction. Firstly, we focus on the events and the mechanisms underlying adhesion, penetration and colonisation of nematodes by Pochonia. We discuss how the infection process is driven by both mechanical forces, induced by the appressoria, and enzymatic activity, specifically by the serine proteases (P32, VCP1, SCP1) and chitinases. Environmental factors have a profound influence on the Pochonia-nematode interaction and these are discussed in detail. Temperature, pH, soil type, soil microbiota and roots can enhance or reduce the parasitism of the nematode by the fungal antagonist. Finally, we discuss how the method of application of Pochonia and its timing can impact on the establishment of the fungus in the soil and, consequently, on the control of nematodes.
Biological control is an alternative to chemical control of plant-parasitic nematodes. This is largely due to public demand for biologically-based and environment-friendly management options for safer pest control. Such demands have had an important impact on biological control research expansion and funding. However, the development of any strain of a biological control agent for nematode control requires many years of research, experimentation, validation and safe-use tests before the biological control agent becomes available to farmers or is further developed by industry as a commercial biopesticide or bionematicide. Biological control potential can be unconstrained when biological control agents are used in combination with compatible integrated pest management tactics, which may include some chemical products and other biological control agent-based products that are currently available on the biopesticide market. This chapter presents part of the history behind some of the initial studies that help to illustrate the scientific work carried out by the many scientists who laid the foundations and helped to develop Pochonia chlamydosporia as a viable, sustainable alternative to chemical control in the integrated management of plant-parasitic nematodes.
THESIS FOR THE OPTION OF THE SCIENTIFIC DEGREE OF DOCTOR IN SCIENCE
Title: Research, develop and innovation of Pochonia chlamydosporia var. catenulata as a microbialagent for the management of root knot nematodes
Author: Agronomist Eng. Leopoldo Hidalgo-Díaz PhD
Date: 16 October 2013
Place: National and Animal Plant Health Centre (CENSA)
ABTRACT
Plant-parasitic nematodes are important pests of many cultivated plants. Vegetable crops account for the greatest proportion of nematicide use because of infection by root-knot nematodes (Meloidogyne spp.). However, the negative impact of most chemical nematicides on both non-target organisms and the environment has led to a total ban or restricted use of these chemicals. Therefore, the development of new control approaches, which can be integrated with other environmentally friendly pest and crop management strategies, is essential. Pochoniachlamydosporiaare among the most promising microbial control agents for nematodes. The research discussed in this thesis concerns the development and innovation of selected native isolates of P.chlamydosporia as amicrobial pest control product for the management of root-knot nematode in vegetable crops in Cuba.The thesis deals with key topics related to these processes and considers the complex interactions present in the practical application of bio-management strategies for Meloidogyne spp. on tropical soils.As a first step a survey was carry out in the two main coffee regions of the country: three species and two varieties of Pochonia spp. were identified from a total of 83 isolates and the strain IMISD 187 of P.chlamydosporia var. catenulate was selected as a potential biological control agent of Meloidogyne spp. A technology for its mass production in solid state fermentation in a polypropylene bag with filter was developed. Itsfollow a good manufacturing practices guide which allows the specific requirements for obtaining the bionematicide KlamiC® to be accomplished. The safe use of the product was demonstrated; it had no adverse ecological effects, indicating its minimum risk to animal and human health and the ecosystem. The basic approach to define a bio-management strategy for organic vegetable production in Cuba is discussed.
Abstract
In the present study influence of pH and temperature on extracellular enzymic activity of protease on solid media were tested of six species of nematode -trapping fungi (Arthrobotrus anchonia , A.dactyloides ,
A.oligospora , Dactylella brochopaga , Dactyleria pyriformic , Monacrosporium eudermatum) . The extracellular enzymic activity of protease were varied according to fungal species and the value of pH and temperature . A.oligospora produced high vegetative growth in all pH values , followed by M.eudermatum , whereas D.pyriformic showed a low vegetative growth followed by A.anchonia . Nevertheless, all tested fungi showed a high vegetative growth at pH = 7.5 , followed by value 7 , whereas it's growth was weak at pH = 8.5 , no vegetative growth showed at pH = 6 . A.oligospora showed a high enzymic activity followed by M.eudermatum , a low enzymic activity was
appeared in D.pyriformic . A best enzymic activity in the tested fungi produced at pH = 7.5 , followed by pH = 7 , while all tested fungi were never showed enzymic activity in pH = 6 and 8.5 . This Study showed that the M.eudermatum produced a highest growth rate in all tested temperature degrees , followed by A.oligospora , whereas D.pyriformic showed lowest growth rate , the vegetative growth in other tested fungi were varied However, the optimum temperature for vegetative growth of all tested fungi was 25 C° , followed by 30 C° , while the lowest vegetative growth was appeared at 40C° followed by 15C° . The high activity of protease showed in A.oligospora , followed by M.eudermatum , the low activity revealed in D.brochopaga, in tested
temperature degrees . the 30C° showed a high enzymic activity . followed by 25C° , on the contrary , no enzymic activity appeared in all tested fungi in both 15C° and 40C° . Keywords: Nematode -trapping fungi , protease , pH , temperature , extracellular enzymic activity
Abstract
The results of the present study revealed that the vegetative growth and sporulation of fungi isolated from freshwater habitat were varied according to fungal species and the used NaCl concentrations. The tow zygomycetes Rhizopus stolonifer and Absidia coerulea produced a high (59 mm , 54 mm for each respectively) vegetative growth in all NaCl concen-trations in comparision with other fungi , whereas Chaetomium sp. produced a low vegetative growth (9 mm) followed by Emericilla nidulans ( 11 mm ) . All tested fungi showed a high vegetative growth in 1 % (60 mm) and 5 % (58mm) NaCl , whereas it`s growth was weak ( 20 mm , 32 mm for each consequantly ) at 0 and 3 % NaCl. The two fungi Bipolaris hawaiinesis and B.spicifera showed an optimum sporulation( more than 3000 spores / ml) in the media supplemented with 0.25%, 0.5% and 1.0% NaCl ,Whereas Ascomycetes fungi were never sporulated in any of the test solutions
Cuticle-degrading-proteases (CDPs) secreted by Beauveria spp. are pivotal biocontrol substances, possessing commercial potential for developing bio-pesticides. Therefore, a thoughtful and contemplative understanding and assessment of the structural and functional features of these proteases would markedly assist the development of biogenic pesticides. Computational molecular biology is a new facile alternative approach to the tedious experimental molecular biology; therefore, by using bioinformatics tools, we isolated and characterized an insect CDP gene from B. bassiana 70 s.l. genomic DNA.
The nematophagous fungus Pochonia chlamydosporia, which belongs to the family Clavicipitaceae (Ascomycota: Pezizomycotina: Sordariomycetes: Hypocreales), is a promising biological control agent for root-knot and cyst nematodes. Its biocontrol effect has been confirmed by pot and field trials. The genome sequence of the fungus was completed recently; therefore, genome-wide functional analyses will identify its infection-associated genes. Gene knockout techniques are useful molecular tools to study gene functions. However, cultures of P. chlamydosporia are resistant to high levels of a range of fungal inhibitors, which makes the gene knockout technique difficult in this fungus. Fortunately, we found that the wild P. chlamydosporia strain PC-170 could not grow on medium containing 150μgml(-1) G418 sulfate, representing a new selectable marker for P. chlamydosporia. The neomycin-resistance gene (neo), which was amplified from the plasmid pKOV21, conferred G418-resistance on the fungus; therefore, it was chosen as the marker gene. We subsequently developed a gene knockout system for P. chlamydosporia using split-marker homologous recombination cassettes with resistance selection and protoplast transformation. The split-marker cassettes were developed using fusion PCR, and involved only two rounds of PCR. The final products comprised two linear constructs. Each construct contained a flanking region of the target gene and two thirds of the neo gene. Alkaline serine protease and chitinase were confirmed to be produced by P. chlamydosporia during infection of nematode eggs and could participate in lysis of the eggshell of nematode eggs. Here, we knocked out one chitinase gene, VFPPC_01099, and two protease genes (VFPPC_10088, VFPPC_06535). We obtained approximately 100 suspected mutants after each transformation. After screening by PCR, the average rate of gene knockout was 13%: 11% (VFPPC_01099), 13% (VFPPC_10088) and 15% (VFPPC_06535). This efficient and convenient technique will accelerate functional genomic studies in P. chlamydosporia.
Copyright © 2014 Elsevier GmbH. All rights reserved.
STABILITY FOR Pochonia chlamydosporia var. catenulata IMI SD STRAIN (KAMYSCHO EX BARRON & ONIONS) ZARE & W. GAMS. PART II. BIOCHEMICAL INDICATOR
ABSTRACT: A fundamental aspect to develop a Biological Control Agent (BCA) is the genetic stability for the selected isolate for mass rearing and its application. Virulence can be attenuated by serial
subcultures. The induction of proteins and different enzymes in liquid medium supplemented with gelatine, chitin or lipid was studied in two IMI SD 187 isolate subcultures, in a time course experiment
during 3, 5 and 7 days. In general the results show there are not significant differences in the behaviour of isolates and there is not an attenuation effect in the enzymatic activity. The gelatine supplemented
medium was a good inductor for all the protein indicators studied. In the chitin medium there was a special induction for VCP 1 protease. The medium with lipids was a good inductor for total proteins,
but in general a bad inductor for the enzymes tested.
The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic, and statistical refinements permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is described for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position Specific Iterated BLAST (PSLBLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities.
Morphological observations and phylogenetic analyses of sequences of small and large ribosomal DNA subunits, the 5.8S and the ITS regions have shown that Verticillium section Prostrata is heterogeneous. Most of its species can be accommodated in the genera Lecanicillium n. gen., comprising the majority of entomogenous or fungicolous species, Pochonia comprising species that parasitize nematode cysts and eggs and often form dictyochlamydospores, and Haptocillium n. gen. comprising nematophagous species with adhesive conidia and sometimes dictyochlamydospores. The genus Rotiferophthora is confirmed as being distinct from Pochonia and Haptocillium. New combinations are introduced for the type species of the three genera. A key to these genera and some similar taxa is provided.
The nematophagous fungus Verticillium chlamydosporium secreted several proteases in submerged culture in which soya peptone was the sole carbon and nitrogen source. One protease, VCP1 (M(r) 33,000, pI 10.2), was purified 14-fold from culture filtrates to apparent homogeneity using preparative isoelectric focusing in free solution, and shown to rapidly hydrolyse the chymotrypsin substrate Suc-(Ala)2-Pro-Phe-pNA and elastin. VCP1 had a Km for Suc-(Ala)2-Pro-Phe-pNA of 4.3 x 10(-5) M and a kcat of 5.8 s-1. It was highly sensitive to PMSF and TPCK, but only moderately sensitive to chicken egg-white and soya bean trypsin inhibitors. VCP1 degraded a wide range of polymeric substrates, including Azocoll, hide protein, elastin, casein and albumin, and accounted for most of the non-specific protease activity detected in culture filtrates. The purified enzyme hydrolysed proteins in situ from the outer layer of the egg shell of the host nematode Meloidogyne incognita and exposed its chitin layer. VCP1 was secreted by several isolates of V. chlamydosporium and V. lecanii, pathogens of nematodes and insects respectively, but not plant-pathogenic species of Verticillium. These observations suggest that VCP1 or similar enzyme(s) may play a role in the infection of invertebrates.
When grown in liquid cultures allowing the formation of nematode traps, the fungus Arthrobotrys oligospora produced two extracellular proteases hydrolysing the chromogenic substrate Azocoll. The protease activity was separated into two fractions (FI and FII) using anion-exchange chromatography. In bioassays, protease(s) present in FII immobilized the free-living nematode Panagrellus redivivus indicating that the enzyme(s) might be involved in the infection of nematodes. A protease designated PII was purified from FII to apparent homogeneity by hydrophobic interaction and size-exclusion chromatography, resulting in an approximately 15-fold increase in specific activity. The purified enzyme was glycosylated, had a molecular mass of approximately 35 kDa (gel filtration) and an isoelectric point of pH 4.6. PII immobilized P. redivivus in bioassays and hydrolysed proteins of the purified cuticle. The enzyme hydrolysed several protein substrates including casein, bovine serum albumin and gelatin, but not native collagen. Examination of substrate specificity with synthetic peptides showed that PII readily hydrolysed tripeptides with aromatic or basic amino acids including N-benzoyl-L-phenylalanyl-L-valyl-L-arginine-4-nitroanilide (Bz-Phe-Val-Arg-NA) and succinyl-glycyl-glycyl-L-phenylalanine-4-nitroanilide (Suc-Gly-Gly-Phe-NA). Mono-peptides were hydrolysed at considerably slower rates. PII had an optimum activity between pH 7 and 9 and was susceptible to autodegradation. PII was inhibited by several serine protease inhibitors including phenylmethylsulfonyl fluoride (PMSF), chymostatin and antipain. The protease was N-terminally blocked, but the sequence of one internal peptide showed a high homology with a region containing the active site histidine residue of the subtilisin family of serine proteases.
Mycoinsecticides are being used for the control of many insect pests as an environmentally acceptable alternative to chemical insecticides. A key aim of much recent work has been to increase the speed of kill and so improve commercial efficacy of these biocontrol agents. This might he achieved by adding insecticidal genes to the fungus, an approach considered to have enormous potential for the improvement of biological pesticides. We report here the development of a genetically improved entomopathogenic fungus. Additional copies of the gene encoding a regulated cuticle-degrading protease (Pr1) from Metarhizium anisopliae were inserted into the genome of M. anisopliae such that Pr1 was constitutively overproduced in the hemolymph of Manduca sexta, activating the prophenoloxidase system. The combined toxic effects of Pr1 and the reaction products of phenoloxidase caused larvae challenged with the engineered fungus to exhibit a 25% reduction in time of death and reduced food consumption by 40% compared to infections by the wild-type fungus. In addition, infected insects were rapidly melanized, and the resulting cadavers were poor substrates for fungal sporulation. Thus, environmental persistence of the genetically engineered fungus is reduced, thereby providing biological containment.
The nematode trapping fungus Arthrobotrys oligospora produces an extracellular serine protease (designated PII) that immobilizes free-living nematodes in bioassays and hydrolyses proteins of the nematode cuticle. Peptides were isolated from PII and partly sequenced. Three internal peptide sequences were used to design synthetic oligonucleotides, which allowed the subsequent isolation of the gene encoding PII from a genomic library. The deduced amino acid sequence indicated that PII is synthesized as a preproenzyme containing the mature enzyme, a signal sequence and a propeptide that are removed before the enzyme is secreted into the medium. The primary sequence of PII displayed a high degree of similarity with several other serine proteases of ascomycetes belonging to the subtilisin family. Northern analysis demonstrated that PII was expressed when the fungus was starved of nitrogen and carbon and that the expression was significantly stimulated by the addition to the medium of various soluble and insoluble proteins, including fragments of nematode cuticle. The levels of the mRNA as well as the proteolytic activity of PII were repressed in the presence of more easily metabolized forms of nitrogen (including ammonia, nitrate and amino acids) or glucose. The activity of the enzyme was almost completely inhibited by the peptide Phe-Val, as well as by the amino acid Phe, without a corresponding decrease in mRNA level. Notably, peptides with similar structures are known to be secreted by the host (nematode) and to stimulate the production of infection structures (traps) of the fungus.
No abstract available.
The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons,
a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST
programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering
the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program
that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining
statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using
this matrix. The resulting Position-Specific Iterated BLAST (PSIBLAST) program runs at approximately the same speed per iteration
as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST
is used to uncover several new and interesting members of the BRCT superfamily.
CLUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W. The new
system is easy to use, providing an integrated system for performing multiple sequence and profile alignments and analysing
the results. CLUSTAL X displays the sequence alignment in a window on the screen. A versatile sequence colouring scheme allows
the user to highlight conserved features in the alignment. Pull-down menus provide all the options required for traditional
multiple sequence and profile alignment. New features include: the ability to cut-and-paste sequences to change the order
of the alignment, selection of a subset of the sequences to be realigned, and selection of a sub-range of the alignment to
be realigned and inserted back into the original alignment. Alignment quality analysis can be performed and low-scoring segments
or exceptional residues can be highlighted. Quality analysis and realignment of selected residue ranges provide the user with
a powerful tool to improve and refine difficult alignments and to trap errors in input sequences. CLUSTAL X has been compiled
on SUN Solaris, IRIX5.3 on Silicon Graphics, Digital UNIX on DECstations, Microsoft Windows (32 bit) for PCs, Linux ELF for
x86 PCs, and Macintosh PowerMac.
Verticillium chlamydosporium is a fungal pathogen of eggs and females of plantparasitic nematodes. The fungus produced an alkaline serine protease in submerged culture. This enzyme, VCPI, was characterized as a class II subtilisin, based on amino acid sequenceh omology. Several of its characteristics, e.g. molecular mass (33 kDa), pI (ca 10) and broad substrate utilisation, are typical of fungal subtilisins. Although some immunological cross-reactivity existed with other enzymes of this class, an antigenic fingerprint was obtained that was distinct, even from the subtilisin that was its closest homologue based on amino acid sequence, PrI from the entomogenous fungus Metarhizium anisopliae.
There was circumstantial evidence, suggesting that this fungal protease was involved in the infection of nematode eggs, which have a largely proteinaceous eggshell. First of all, the enzyme was able to remove the outer protein layer from eggs of the susceptible root-knot nematode, Meloidogyne incognita, exposing the underlying chitin layer. Scanning electron microscopy revealed that fungal hyphae on the egg surface left an imprint, presumably through enzymatic action. There was also evidence of the protease weakening the eggshell, as enzyme-treated nematode eggs were more easily lysed and infected by the fungus than those not pre-incubated in the enzyme. A polyclonal antibody against VCPI demonstrated protease production by the fungus, prior to, or concurrent with, penetration. The enzyme was associated with appressoria, i.e. fungal infection structures. In contrast to the susceptible root-knot nematode, VCPI had little impact on the egg shell of the potato cyst nematode Globodera rostochiensis. It is suggested that the limited in situ hydrolysis of G. rostochiensis egg shell proteins is a factor contributing to its relative resistance to the fungus.
Regulation studies in batch culture showed that production of the protease VCPI was repressed by high carbon and nitrogen levels. Its basic regulatory mechanism was that of repression/derepression. However, the highest protease titre was obtained when M incognita eggs were present in the medium, suggesting induction by the host. Collagen and chitin were possibly responsible for this inductive effect.
In conclusion, it is believed that VCPI is a protease with a dual role for V chlamydosporium. During saprotrophic growth, VCP1 would allow the fungus to scavenge nutrients from a wide range of protein sources. However, the enzyme also has a designated function in penetration of the host, which makes it a versatile tool for a fungus that can switch trophic modes during its life-cycle.
The achievements of this research include the first demonstration in a nematode-attacking fungus of:
-a well-characterized protease, including data on stability, kinetics and
isoforms;
-a subtilisin-like protease in an egg-parasitic nematophagous fungus;
-a pathogenicity-related enzyme in V chlamydosporium;
-a determinant of host specificity;
- enzyme regulation in general, and induction by the host, in particular.
The proteinaceous insect cuticle is an effective barrier against most microbes, but entomopathogenic fungi can breach it using extracellular proteases. We report here the isolation and characterization of a cDNA clone of the cuticle-degrading protease (Pr1) of Metarhizium anisopliae. The cDNA sequence revealed that Pr1 is synthesized as a large precursor (40.3 kDa) containing a signal peptide, a propeptide and the mature protein predicted to have a molecular mass of 28.6 kDa. The primary structure of Pr1 has extensive similarity with enzymes of the subtilisin subclass of serine endopeptidases and the serine, histidine and aspartate components of the active site in subtilisins are preserved. Proteinase K demonstrated the closest sequence similarity to Pr1 (61%) but Pr1 was twofold more effective than proteinase K at degrading isolated cuticles of Manduca sexta and 33-fold more effective at degrading structural proteins bound to the cuticle by covalent bonds. We postulate that the additional positively charged residues on the surface of the Pr1 molecule, as determined using proteinase K, may facilitate electrostatic binding to cuticle proteins which is a prerequisite for activity. Northern-blot analysis of RNA and nuclear run-on assays demonstrated transcriptional control of the expression of Pr1 during nutrient deprivation and during the formation of infection structures. Southern-blot analysis demonstrated that genes with significant homologies to Metarhizium Pr1 were present in the entomopathogens Aspergillus flavus and Verticillium lecanii but not Zoophthora (= Erynia) radicans.
The factors which are involved in the in vitro adsorption of Metarhizium anisopliae, Verticillium lecanii, and Beauveria bassiana proteases onto insect cuticle and the hydrolysis of cuticle proteins were investigated. Several proteases are produced by these fungi during growth in liquid media containing 1% (w/v) ground grasshopper cuticle, and the most basic of these proteases adsorb onto cuticle. Protease adsorption was maximum in a broad pH range from 4 to 7. Adsorption was also time dependent, with maximum adsorption occurring within 1 hr. Up to 50% of the adsorbed protease could be dissociated from the cuticle with 10 mM potassium phosphate (pH 7) but not with distilled water. Lysine and glutamic acid interfered with protease adsorption, while proline, alanine, and tyrosine did not. Modification of cuticle protein carboxyl groups by esterification or by carboxamidation resulted in a decrease of protease adsorption. Acetylation of the cuticle tyrosine hydroxyl groups resulted in decreased adsorption of M. anisopliae and V. lecanii protease but not B. bassiana protease. Phenylglyoxylation masked the amino groups with carboxyl groups and this resulted in increased protease adsorption. An increase in protease adsorption onto chemically modified cuticles was not necessarily correlated with increased hydrolysis. Results suggested that the proteases produced by M. anisopliae and V. lecanii were similar in their adsorption properties. All proteases required a carboxyl group for adsorption but M. anisopliae and V. lecanii proteases also required an unsubstituted hydroxyl group with a carboxyl group for maximum adsorption.
The infection process of the nematophagous fungus Verticillium chlamydosporium (isolate Vc1O) was studied in two nematode species that differed in susceptibility according to standardized in vitro assays. After 14 d, V. chlamydosporium infected 9 and 80% of Globodera rostochiensis and Meloidogyne incognita eggs, respectively. The purified proteinase VCPl, from V. chlamydosporium, removed the protein coat from eggs of M. incognita but not of G. rostochiensis. VCPl was more effective at hydrolysing proteins from M. incognita eggs than the related enzymes Prl and proteinase K, whereas the latter two were slightly more active against G. rostochiensis eggs than VCPl. Pretreatment of eggs with VCPl increased susceptibility to V. chlamydosporium from 80–100% in M. incognita eggs and from 9 to 29% in G. rostochiensis eggs. These observations suggest that VCPl is a host specificity-determining factor and also confirm it to be a pathogenicity determinant which works best in concert with other enzymes secreted by the actively growing fungus. Fungal growth on the surface of eggs of both species was commensurate with the infection frequencies observed; growth was limited and fewer appressoria were observed on eggs of G. rostochiensis compared with those of M. incognita. Eggs of M. incognita denatured by heat or killed with sodium azide, like eggs treated with VCPl, were more susceptible than untreated, healthy eggs. In contrast heat and azide treatment of G. rostochiensis eggs did not increase the number of infections. Appressoria were observed on the eggs of both nematode species in all treatments, but the numbers and types varied greatly. On M. incognita, the dominant type was swollen and branched laterally from hyphae, while on G. rostochiensis lateral inconspicuous appressoria prevailed. Differentiation of appressoria on live and dead nematode eggs showed that their production was not limited to the parasitic state of the fungus.
Les champignons entomopathogenes et phytopathogenes du genre Verticillium sont doues d'une capacite d'adaptation enzymatique (production et regulation) permettant la degradation de certains polymeres contenus dans le tegument de leurs hotes respectifs. Par exemple, les phytopathogenes facultatifs (V. albo-atrum et V. dahliae) produisaient de plus grandes quantites de cellulase et de xylanase que l'entomopathogene facultatif (V. lecanii). Le V. lecanii produisait une protease extracellulaire de type subtilisine durant sa croissance dans un milieu contenant des cuticules d'insectes mais pas dans un milieu contenant des parois de cellules vegetales et le phytopathogene V. alboatrum formait un compose de capacite reduite de regulation de la production de cette enzyme. Les pathogenes opportunistes (V fungicola et V. coccosporum) et l'espece saprophyte (V. rexianum) etaient moins specifiques au niveau de la regulation et de la production de certaines proteases ainsi que des cellulases et des xylanases. Un dendrogramme construit d'apres l'analyse de groupes des profils obtenus par API-ZYM a revele des normalisations d'un large eventail d'enzymes extracellulaires a l'interieur d'un groupe hote-pathogene (i.e. pathogene des insectes ou des plantes). Les pathogenes opportunistes etaient disperses dans le dendrogramme probablement a cause de la diversite du type et de l'expression des enzymes extracellulaires.
The insect and plant pathogens within the fungal genus Verticillium showed enzymatic adaptation (production and regulation) directed to the degradation of some of the polymers found in the integument of their respective hosts. For example, the facultative plant pathogens (V. albo-atrum and V. dahliae) produced greater levels of cellulase and xylanase than the facultative insect pathogen (V. lecanii). Verticillium lecanii produced extracellular subtilisin-like protease when grown in insect cuticle medium but not in plant cell wall medium, while the plant pathogen V. albo-atrum showed a diminished regulatory component in the production of this enzyme. The opportunistic pathogens (V. fungicola and V. coccosporum) and the saprobic species (V. rexianum) were less specific in the production and regulation of several proteases as well as cellulases and xylanases. A dendrogram based on cluster analysis compiled from fungal API-ZYM profiles showed commonalties in a broad array of extracellular enzymes within a host-pathogen group (i.e. insect or plant pathogen). The opportunistic pathogens were dispersed throughout the dendrogram, suggestive of the diversity in type and expression of extracellular enzymes.Key words: extracellular enzymes, pathogenic fungi.
The proteinaceous outer integument of insects forms an effective barrier against most microbes. Only the 700 known species of entomopathogenic fungi effect entry into their hosts by breaching the cuticle. There is accumulating evidence that the ability of fungi to degrade protein may aid their invasion of and growth in this orderly complex structure. Evidence for the particular importance of proteinases derives largely from studies of their production in infected cuticles associated with cuticle degradation, the effects of proteinase inhibitors on pathogen behavior, and by the analysis of protease-deficient mutants. More recently, studies have included the cloning, identification, and manipulation of specific protease genes of Metarhizium anisopliae, particularly those of the subtilisin (chymoelastase) type (designated Pr1) also produced by many other entomopathogenic fungi. Following solubilization of cuticle proteins by Pr1-type endoproteases, complete degradation of the cuticle involves a number of interacting enzyme species including a family of trypsin-like proteinases, metalloproteinases, several aminopeptidases, and a carboxypeptidase. Testing genetically engineered M. anisopliae null mutants of Pr1 indicated that the other endopeptidases can partially substitute for Pr1. The exopeptidases further degrade peptides released by the endopeptidases producing free amino acids for uptake and metabolism. Utilization of these enzymes has assisted investigators in understanding cuticle structure and how the cuticle is degraded naturally, and could lead to improved strain selection of entomopathogenic fungi or the introduction of their genes into other microbes and plants for the purpose of insect control. Key words: proteinaceous insect cuticle, pathogen endopeptidases, exopeptidases, multiple isozymes, enzyme regulation.
Most populations of the cereal cyst-nematode are parasitised by fungi killing females and eggs. The life history of an Entomophthora-like fungus, which kills females and causes the breakdown of the cuticle preventing cyst formation, is described. This fungus attacks other cyst-nematodes but not Globodera rostochiensis. A method is described for estimating the number of diseased eggs in females and cysts. Verticillium chlamydosporium was the most frequently observed egg parasite; it killed 50% of the eggs in females on barley roots in July in a field trial. Tarichium auxiliare and Cylindrocarpon destructans were found more frequently in soils where the nematode failed to multiply than in those where it increased but were not considered important as few females were infected. Similar numbers of new females were produced in soils where populations multiply or decline but in decline soils many failed to form cysts and few healthy eggs were produced.
The fine structure of egg shells of four different genera belonging to the order Tylenchida has been examined. The species examined were Meloidogyne javanica, Rotylenchulus reniformis, Tylenchulus semipenetrans and Pratylenchus minyus . They are all similar in their basic structure, being composed of vitelline membrane, chitin and lipid layers, but there is considerable variability in the thickness of these layers.
We have retained the conventional nomenclature because of its convenience, but it is clear that these layers have a variety of chemical components. However, they do appear to contain the compounds from which they take their name. Thus chitin occurs in the chitin layer, and lipid in the lipid layer. The latter is removed by the technique used in isolating the shell from the egg. Chemical analysis of the hydrolysis products of these shells has revealed a high (35 %) proline content which appears to be a characteristic of those nematode egg shells which have been examined so far. These analyses and treatment with enzymes indicate that the chitin layer is a chitin–protein complex.
Experiments on the permeability of eggs of M. javanica at different temperatures indicate that changes in permeability are not due to the melting of a single lipid with a distinct melting point as had been thought in the past. We have found that Arrhenius activation energies calculated from the two slopes of an Arrhenius plot were 17·8 kcal/mol and 43·0 kcal/mol respectively, the transition from one to the other taking place at 62°C. We think that these changes are due to changes in the properties of lipoprotein membranes in the lipid layer. These membranes appear to be of paramount importance in controlling the permeability of the nematode egg shell.
Populations (eggs/g dried soil) of the cereal cyst nematode Heterodera avenae Woll. failed to increase at two sites on susceptible spring barley but increased slightly at another on spring oats. Nematode multiplication was not related to the numbers of females produced on roots in summer; fewer female nematodes were produced where populations increased than where they declined. The failure of the nematode to multiply was due to few (less than 40%) of the females forming cysts containing eggs, a reduction in fecundity and an increase in the number of encysted eggs containing fungi. Verticillium chlamydosporium was the main fungus causing the decline in nematode numbers. Nematophthora gynophila was present at all sites but was most active at Woburn where irrigation was used to keep soils moist. V. chlamydosporium was the main parasite of encysted eggs at all sites but soil-borne saprophytic fungi were also isolated. These fungi were not found in many eggs and were considered to be only weak parasites. Differences in resting fungal spore densities in soil were not always related to differences in rates of parasitism. The activity of N. gynophila is affected by soil moisture and density of females as well as the density of spores in soil. The production of chlamydospores by V. chlamydosporium may also depend on the isolate of fungus present: some isolates produce no such spores on artificial media or, possibly, in soil. This work has shown that V. chlamydosporium and N. gynophila can reduce cereal cyst nematode populations under susceptible cereals but it is difficult to predict at present if they will give an effective control in a given soil or season.
Fluorescein-labeled anti-rabbit antiserum showed that a serine protease designated P32, produced by the nematophagous fungusVerticillium suchlasporium, is secreted during infection of nematode eggs. Increased fluorescence in appressoria of the fungus on eggshells of the plant parasitic nematodeHeterodera schachtii indicated the presence of P32 in these fungal structures. Appressoria are involved in host penetration and these results support a role for P32 in the pathogenicity of the fungus to nematode eggs. These findings agree with similar results of entomogenous fungi penetrating the insect cuticle.
Abstract A Beauveria bassiana extracellular subtilisin-like serine endoprotease is a potential virulence factor by virtue of its activity against insect cuticles. A cDNA clone of the protease was isolated from mycelia of B. bassiana grown on cuticle/chitin cultures. The amino acid sequence of this gene was compared to that of Metarhizium anisopliae Pr1, the only pathogenicity determinant so far described from an entomopathogenic fungus, and proteinase K, isolated from Tritirachium album, a saprophytic fungus. The cDNA sequence revealed that B. bassiana Prl is synthesized as a large precursor (Mr 37 460) containing a signal peptide, a propeptide and the mature protein predicted to have an Mr of 26 832.
Structure-based mutational analysis of serine protease specificity has produced a large database of information useful in addressing biological function and in establishing a basis for targeted design efforts. Critical issues examined include the function of water molecules in providing strength and specificity of binding, the extent to which binding subsites are interdependent, and the roles of polypeptide chain flexibility and distal structural elements in contributing to specificity profiles. The studies also provide a foundation for exploring why specificity modification can be either straightforward or complex, depending on the particular system.
Subtilases are members of the clan (or superfamily) of subtilisin-like serine proteases. Over 200 subtilases are presently known, more than 170 of which with their complete amino acid sequence. In this update of our previous overview (Siezen RJ, de Vos WM, Leunissen JAM, Dijkstra BW, 1991, Protein Eng 4:719–731), details of more than 100 new subtilases discovered in the past five years are summarized, and amino acid sequences of their catalytic domains are compared in a multiple sequence alignment. Based on sequence homology, a subdivision into six families is proposed. Highly conserved residues of the catalytic domain are identified, as are large or unusual deletions and insertions. Predictions have been updated for Ca2+-binding sites, disulfide bonds, and substrate specificity, based on both sequence alignment and three-dimensional homology modeling.
We report the isolation of cDNA clones containing the full 6.3-kb coding sequence of thepara-type sodium channel gene of the housefly,Musca domestica. This gene has been implicated as the site of knockdown resistance (kdr), an important resistance mechanism that confers nerve insensitivity to DDT and pyrethroid insecticides. The cDNAs predict a polypeptide of 2108 amino acids with close sequence homology (92% identity) to theDrosophila para sodium channel, and around 50% homology to vertebrate sodium channels. Only one major splice form of the housefly sodium channel was detected, in contrast to theDrosophila para transcript which has been reported to undergo extensive alternative splicing. Comparative sequence analysis of housefly strains carryingkdr or the more potentsuper-kdr factor revealed two amino acid mutations that correlate with these resistance phenotypes. Both mutations are located in domain II of the sodium channel. A leucine to phenylalanine replacement in the hydrophobic IIS6 transmembrane segment was found in two independentkdr strains and sixsuper-kdr strains of diverse geographic origin, while an additional methionine to threonine replacement within the intracellular IIS4-S5 loop was found only in thesuper-kdr strains. Neither mutation was present in five pyrethroid-sensitive strains. The mutations suggest a binding site for pyrethroids at the intracellular mouth of the channel pore in a region known to be important for channel inactivation.
The pr1 gene of the entomopathogenic fungus Metarhizium anisopliae encodes a serine protease that is highly active towards the insect cuticle and whose synthesis is subject to both carbon
and nitrogen repression. The pr1 promoter region was sequenced revealing the presence of putative CREA- and AREA-binding sites. In vitro bandshift experiments
demonstrated that an Aspergillus nidulans GST-CREA fusion protein was capable of binding to two of the three putative CREA sites. Using a PCR-based strategy the M. anisopliae crr1 gene was identified; it encodes a putative C2H2-type DNA-binding protein with significant sequence similarity to A. nidulans CREA. Complementation experiments with an A. nidulans strain carrying creA204 demonstrated that CRR1 can partially substitute for CREA function.
The extracellular subtilisin-like protease activity of several fungal pathogens of nematodes, insects and plants was recorded after batch cultivation. Nematophagous and entomophagous isolates of Verticillium, Paecilomyces, Beauveria and Metarhizium produced Suc-(Ala)2-Pro-Phe-pNA hydrolysing enzymes that differed in serological properties based on Western blot analysis with polyclonal antibodies. The antibodies used were raised against the main subtilisin-like proteases from one species from each of the four genera. From each strain of V. chlamydosporium tested, between one and four isoforms with different pI values were separated by isoelectric focusing and visualized by enzymoblots. The occurrence of multiple isoforms with unique N-terminal sequences in single strains might suggest the presence of subtilisin gene families. Using as a probe a fragment of the Pr1 gene from M. anisopliae, RFLPs were observed that confirmed qualitative differences between subtilisin-like genes within and between species. In contrast to these pathogens of invertebrates, plant-pathogenic species of Verticillium did not produce subtilisin-like proteases, nor were homologous genes detected. Such activity, however, and the corresponding genes, were detected in species weakly pathogenic to plants, or saprotrophs.
Almost every property of subtilisin, a simple bacterial serine protease, has been altered by protein engineering including its catalysis, substrate specificity, pH/rate profile, and stability to oxidative, thermal and alkaline inactivation.
Beauveria bassiana extracellular protease was shown to hydrolyze protein in cuticle of the migratory grasshopper, Melanoplus sanguinipes. Protease-proficient and protease-deficient strains of B. bassiana were assessed for production of extracellular protease in liquid culture and their virulence, as measured by LT50, toward M. sanguinipes. A uv irradiation-derived protease-deficient mutant strain produced less than 10% of protease when compared to the protease-proficient strains when grown in 1% (w/v) gelatin liquid medium or 1% (w/v) ground M. sanguinipes cuticle liquid medium. The protease-proficient strains showed the lowest LT50 (5.71–6.80 days) against the migratory grasshopper, while the protease-deficient strain had the highest LT50 (11.34 days). There was a negative relationship between protease production after 2 days of growth in liquid culture by the various strains and their associated LT50. We conclude that, in this study, protease production by the B. bassiana protease-deficient strain was low enough so that it extended the time course of pathogenesis toward M. sanguinipes.
PCR-based assays were performed to resolve the genetic variation between 28 different isolates of Verticillium chlamydosporium using primers designed to amplify ribosomal internal transcribed spacers (ITS) and intergenic spacers (IGS). Different isolates of V. chlamydosporium were also differentiated using primers matching enterobacterial repetitive intergenic consensus (ERIC) sequences and repetitive extragenic palindromic (REP) elements. Restriction fingerprinting of PCR-amplified ITS products failed to yield intraspecific polymorphism, and different levels of discrimination between V. chlamydosporium isolates were not achieved. However restriction patterns of ITS products digested with Hae III and Hinf I were useful in differentiating between some of the closely related isolates of V. chlamydosporium, plant pathogenic Verticillium species and some common soil fungi. PCR amplification of IGS was found to be the most sensitive method which enabled the detection of 22 variants within the sample of 28 isolates of V. chlamydosporium and six different plant pathogenic Verticillium species. By using ERIC and REP-PCR fingerprinting, isolates were categorized in 20 and 13 genotypes, respectively. In general, PCR-based procedures can differentiate between closely related isolates of V. chlamydosporium within IGS genotypes. This also could be achieved by ERIC and REP-PCR, and may be considered a rapid tool for the genetic characterization and detection of different isolates of V. chlamydosporium.
Subtilases are members of the family of subtilisin-like serine proteases. Presently, >50 subtilases are known, >40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase and proteinase K). The mature enzymes contain up to 1775 residues, with N-terminal catalytic domains ranging from 268 to 511 residues, and signal and/or activation-peptides ranging from 27 to 280 residues. Several members contain C-terminal extensions, relative to the subtilisins, which display additional properties such as sequence repeats, processing sites and membrane anchor segments. Multiple sequence alignment of the N-terminal catalytic domains allows the definition of two main classes of subtilases. A structurally conserved framework of 191 core residues has been defined from a comparison of the four known three-dimensional structures. Eighteen of these core residues are highly conserved, nine of which are glycines. While the α-helix and β-sheet secondary structure elements show considerable sequence homology, this is less so for peptide loops that connect the core secondary structure elements. These loops can vary in length by >150 residues. While the core three-dimensional structure is conserved, insertions and deletions are preferentially confined to surface loops. From the known three-dimensional structures various predictions are made for the other subtilases concerning essential conserved residues, allowable amino acid substitutions, disulphide bonds, Ca2+-binding sites, substrate-binding site residues, ionic and aromatic interactions, proteolytically susceptible surface loops, etc. These predictions form a basis for protein engineering of members of the subtilase family, for which no three-dimensional structure is known.
The genomic DNA for the alkaline protease (Alp) of the fungus Aspergillus oryzae was isolated using synthetic oligonucleotides as hydridization probes, and the complete nucleotide sequence was identified. The Alp gene is 1374 nucleotides long and contains three introns, one of which is in the pro region and two in the mature coding region. Sequences related to the TATA box (TATAAAT) and the CAAT box (CCAAAT) were found in the 5'-noncoding region. Primer extension analysis showed that three transcriptional start points are present.
Complementary DNA encoding Ac. chrysogenum alkaline protease (Alp) was isolated from the Ac. chrysogenum ATCC11550 cDNA library by express-blot assay. The genomic DNAs encoding Ac. chrysogenum Alp were isolated from the Ac. chrysogenum genomic DNA library using the cloned cDNA as a probe. The 3150 nucleotides of the gene were sequenced. The prepro-Alp consists of 402 amino acids and two intervening sequences are found within the coding region. The amino acid sequence of Ac. chrysogenum Alp has 57% homology to that of Aspergillus oryzae Alp. The entire cDNA, encoding Ac. chrysogenum Alp, when introducing into the yeast Saccharomyces cerevisiae, directed the secretion of enzymatically active Alp into the culture medium.
Elastase, a potential virulence factor from the opportunistic pathogen Aspergillus fumigatus, was purified 220-fold from culture broth by fast-performance liquid chromatography employing anion exchange (Q Sepharose fast flow), cation exchange (S Sepharose fast flow), and gel filtration (Superose 12). Purified to near homogeneity, the elastase had an apparent molecular mass of 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver stain) but a mass of about 19.1 kDa as determined by gel filtration on Superdex 75. The elastase is not glycosylated and is positively charged at neutral pH, having a pI of 8.75. Inhibition by 0.2 mM phenylmethylsulfonyl fluoride (100%) and 0.21 mM leupeptin (60%) implies that the elastase is a serine protease. However, the enzyme is also inhibited by 5 mM EDTA (100%) and 10 mM 1,10-orthophenanthroline (30%), suggesting a requirement for divalent cations. The enzyme acts optimally at pH 7.4 and 45 degrees C in 50 mM sodium borate buffer, but in Tris HCl, the pH optimum shifts to 8.8.
The proteinaceous insect cuticle is an effective barrier against most microbes, but entomopathogenic fungi can breach it using extracellular proteases. We report here the isolation and characterization of a cDNA clone of the cuticle-degrading protease (Pr1) of Metarhizium anisopliae. The cDNA sequence revealed that Pr1 is synthesized as a large precursor (40.3 kDa) containing a signal peptide, a propeptide and the mature protein predicted to have a molecular mass of 28.6 kDa. The primary structure of Pr1 has extensive similarity with enzymes of the subtilisin subclass of serine endopeptidases and the serine, histidine and aspartate components of the active site in subtilisins are preserved. Proteinase K demonstrated the closest sequence similarity to Pr1 (61%) but Pr1 was twofold more effective than proteinase K at degrading isolated cuticles of Manduca sexta and 33-fold more effective at degrading structural proteins bound to the cuticle by covalent bonds. We postulate that the additional positively charged residues on the surface of the Pr1 molecule, as determined using proteinase K, may facilitate electrostatic binding to cuticle proteins which is a prerequisite for activity. Northern-blot analysis of RNA and nuclear run-on assays demonstrated transcriptional control of the expression of Pr1 during nutrient deprivation and during the formation of infection structures. Southern-blot analysis demonstrated that genes with significant homologies to Metarhizium Pr1 were present in the entomopathogens Aspergillus flavus and Verticillium lecanii but not Zoophthora (= Erynia) radicans.
The cDNA and the chromosomal gene encoding proteinase K from Tritirachium album Limber have been cloned in Escherichia coli and the entire nucleotide sequences of the coding region, as well as 5′- and 3′-flanking regions have been determined. The deduced primary translation product consisting of 384 amino acid residues (molecular mass = 40 231 Da) contains an N-terminal region of 105 amino acids not present in the mature protein. By analogy to the evolutionary-related bacterial subtilisins and other serine proteinases it is inferred that the primary secreted product is a zymogen containing a 15-amino-acid signal sequence and a 90-amino-acid propeptide. The propeptide is presumably removed in the later steps of the secretion process or upon secretion into the medium.
The nucleotide-sequence analysis of the gene and its flanking regions has revealed that the proteinase-K gene is composed of two exons and one 63-bp-long intron located in the proregion. Furthermore, a putative promoter sequence and a capping site have been identified, suggesting that the transcription-start site is located 103-bp upstream of the ATG initiation codon. To express the proproteinase-K gene in E. coli, proproteinase-K cDNA was cloned in a plasmid vector under control of the tac promoter. The hybrid plasmid pSPPRO, constructed for this purpose, contained the cDNA coding for proproteinase K [from Ala (-91) to the C-terminal Ala (279)] fused to the N-terminal-signal-peptide sequence of the alkaline-phosphatase gene preceded by the tac promoter. E. coli BMH71-18, harbouring this plasmid, exhibited slight proteolytic activity when tested on skimmed-milk plates, suggesting that some fusion proteins were correctly secreted into the periplasm and processed to the mature proteinase K.
Two chymoelastases and three trypsinlike proteases were separated from culture filtrates of the entomopathogen Metarhizium anisopliae. A chymoelastase (Pr1) (pI 10.3 Mr 25,000) and trypsin (Pr2) (pI 4.42, Mr 28,500) were purified to homogeneity by ammonium sulphate precipitation, isoelectric focusing, and affinity chromatography. Inhibition studies showed that both enzymes possessed essential serine and histidine residues in the active site. Pr1 shows greater activity than Pr2 or mammalian enzymes against locust cuticle and also possesses activity vs elastin. Pr1 shows a broad primary specificity toward amino acids with hydrophobic side groups in synthetic ester and amide substrates. The kinetic properties of Pr1 demonstrate a preference for extended peptide chains with the active site recognising at least five substrate residues. The S5 and S4 subsites show a preference for negatively charged succinyl and hydrophobic acetyl groups, respectively. The S3 and S2 subsites both discriminated in favor of alanine and against proline. Pr2 rapidly hydrolyzed casein and synthetic substrates containing arginine or lysine. It possessed little or no activity vs cuticle, elastin, or synthetic substrates for chymotrypsin and elastase. Specific active site inhibitors confirmed the similarities between Pr2 and trypsin.
The major extracellular proteases from the nematophagous fungus Verticillium chlamydosporium and the entomophagous fungus Metarhizium anisopliae, VCP1 and Pr1, respectively, are closely related both functionally and serologically. Antibodies raised against either enzyme cross-reacted with both antigens, suggesting that they have common epitopes. The VCP1 and Pr1 antisera labelled bovine pancreatic elastase and proteinase K, respectively. Neither antiserum reacted with commercial chymotrypsin. An antiserum to a serine protease from the closely related V. suchlasporium also cross-reacted with VCP1 and Pr1. In contrast, a polyclonal antibody to an isoform of Pr1 exclusive to M. anisopliae isolate ME1 failed to recognize Pr1 from M. anisopliae V245 or VCP1. The N-terminal amino acid sequence of VCP1 revealed similarities with subtilisin-like enzymes from other fungi, but the closest match was with Pr1. The pure enzymes, VCP1 and Pr1, failed to hydrolyse mono-aminoacyl-naphthylamide substrates but demonstrated dipeptidyl peptidase activity against Gly-Pro-beta NA and Leu-Ala-beta NA, respectively. These results are discussed in the context of specificity of invertebrate mycopathogens.
We have cloned a genomic DNA encoding the alkaline protease (Alp) of Fusarium sp. S-19-5 from a genomic DNA library and sequenced the nucleotides. Complementary DNA encoding Alp was also isolated from the cDNA library after amplifying the gene by PCR using partial sequences of the Alp genomic DNA as primers. The Alp gene has an open reading frame of 1137 nucleotides containing three introns. A TATA box (TAAATA) was observed 112 base pairs upstream from the translation initiation codon in the 5'-non coding region. The Alp protein has a pre region consisting of 14 amino acids and a pro region of 85 amino acids preceding the mature region, which consists of 280 amino acids. The amino acid sequence of Fusarium Alp has 52% homology with that of Aspergillus oryzae and 51% homology with that of Acremonium chrysogenum. The entire cDNA encoding Fusarium Alp was introduced into Saccharomyces cerevisiae, which then secreted enzymatically active Alp into the culture medium.
A Beauveria bassiana extracellular subtilisin-like serine endoprotease is a potential virulence factor by virtue of its activity against insect cuticles. A cDNA clone of the protease was isolated from mycelia of B. bassiana grown on cuticle/chitin cultures. The amino acid sequence of this gene was compared to that of Metarhizium anisopliae Pr1, the only pathogenicity determinant so far described from an entomopathogenic fungus, and proteinase K, isolated from Tritirachium album, a saprophytic fungus. The cDNA sequence revealed that B. bassiana Pr1 is synthesized as a large precursor (M(r) 37,460) containing a signal peptide, a propeptide and the mature protein predicted to have an M(r) of 26,832.
Entomopathogenic fungi are attracting attention as potential biological control agents of insect pests. The mechanisms of pathogenesis have parallels with those of some plant-pathogenic fungi, particularly in the areas of formation of an infection structure, entry into the host and toxin-mediated host death. Understanding these processes will provide a rational basis for strain selection and improvement.
Subtilases are members of the family of subtilisin-like serine proteases. Over 70 subtilases are presently known, more than 60 of which with their complete amino acid sequence. In this update, details of more than 20 new subtilases discovered recently are summarized, and their sequences included in a multiple sequence alignment table.
I describe the current version of the sequence analysis package developed at the MRC Laboratory of Molecular Biology, which has come to be known as the "Staden Package." The package covers most of the standard sequence analysis tasks such as restriction site searching, translation, pattern searching, comparison, gene finding, and secondary structure prediction, and provides powerful tools for DNA sequence determination. Currently the programs are only available for computers running the UNIX operating system. Detailed information about the package is available from our WWW site: http:@www.mrc-lmb.cam.ac.uk/pubseq/.
A range of specialist and generalist microorganisms in the rhizosphere attacks plant-parasitic nematodes. Plants have a profound effect on the impact of this microflora on the regulation of nematode populations by influencing both the dynamics of the nematode host and the structure and dynamics of the community of antagonists and parasites in the rhizosphere. In general, those organisms that have a saprophytic phase in their life cycle are most affected by environmental conditions in the rhizosphere, but effects on obligate parasites have also been recorded. Although nematodes influence the colonization of roots by pathogenic and beneficial microorganisms, little is known of such interactions with the natural enemies of nematodes in the rhizosphere. As nematodes influence the quantity and quality of root exudates, they are likely to affect the physiology of those microorganisms in the rhizosphere; such changes may be used as signals for nematode antagonists and parasites. Successful biological control strategies will depend on a thorough understanding of these interactions at the population, organismal, and molecular scale.
Ver-ticillium chlamydosporium, fungal parasite of Meloidogyne arenaria females
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Isolation of un-known flanking DNA by a simple two-step polymerase chain re-action method. Dynabeads 1 M-280 Streptavidin (Prod. No. 112.05/ 06 and 602.10) applications Character-ization of cuticle-degrading proteases produced by the en-tomopathogen Metarhizium anisopliae
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Isolation of unknown flanking DNA by a simple two-step polymerase chain reaction method. Dynabeads ® M-280 Streptavidin (Prod
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Variation in the subtilisins of fungal pathogens of insects and nematodes
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