Plant Pathology

The veA or velvet gene is necessary for biosynthesis of mycotoxins and other secondary metabolites in Aspergillus species. In addition, veA has also been demonstrated to be necessary for normal seed colonization in Aspergillus flavus and Aspergillus parasiticus. The present study shows that veA homologues are broadly distributed in fungi, particularly in Ascomycetes. The Fusarium verticillioides veA orthologue, FvVE1, is also required for the synthesis of several secondary metabolites, including fumonisin and fusarins. This study also shows that maize plants grown from seeds inoculated with FvVE1 deletion mutants did not show disease symptoms, while plants grown from seeds inoculated with the F. verticillioides wildtype and complementation strains clearly showed disease symptoms under the same experimental conditions. In this latter case, the presence of lesions coincided with accumulation of fumonisins in the plant tissues, and only these plant tissues had elevated levels of sphingoid bases and their 1-phosphate derivatives, indicating inhibition of ceramide synthase and disruption of sphingolipid metabolism. The results strongly suggest that FvVE1 is necessary for pathogenicity by F. verticillioides against maize seedlings. The conservation of veA homologues among ascomycetes suggests that veA could play a pivotal role in regulating secondary metabolism and associated pathogenicity in other fungi.

A looming challenge for agriculture is sustainable intensification of food production to feed the growing human population. Current chemical and genetic technologies used to manage plant diseases are highly vulnerable to pathogen evolution and are not sustainable. Pathogen evolution is facilitated by the genetic uniformity underlying modern agroecosystems, suggesting that one path to sustainable disease control lies through increasing genetic diversity at the field scale by using genetically diverse host mixtures. We investigate how host mixtures can improve disease control using a population dynamics model. We find that when a population of crop plants is exposed to host-specialized pathogen species or strains, the overall disease severity is smaller in the mixture of two host varieties than in each of the corresponding pure stands. The disease severity can be minimized over a range of mixing ratios. These findings may help in designing host mixtures that efficiently control diseases of crops. We then generalize the model to describe host mixtures with many components. We find that when pathogens exhibit host specialization, the overall disease severity decreases with the number of components in the mixture. As the degree of specialization increases, the decrease in disease severity becomes larger. Using these model outcomes, we propose ways to optimize the use of host mixtures to decrease disease in agroecosystems.

Benzothiadiazole (BTH), as Bion WG50, and acetylsalicylic acid (ASA) treatments of potato foliage of field- and glasshouse-grown potato plants were compared for control of two foliar diseases, early blight (Alternaria solani) and powdery mildew (Erysiphe cichoracearum). The effect of these treatments on harvested tubers wound-inoculated with the dry rot fungus (Fusarium semitectum) was also evaluated. BTH (50 mg a.i. L−1) gave almost complete control of both foliar pathogens on inoculated glasshouse-grown plants and reduced the severity of leaf spotting diseases (mainly early blight) in the field. BTH (100 mg a.i. L−1) and ASA (400 mg a.i. L−1) reduced the severity of dry rot in field-grown tubers in some post-harvest wound-inoculated treatments but not others and a similar reduction occurred with tubers inoculated post-harvest from BTH-treated plants grown under glasshouse conditions. BTH treatment increased β-1,3-glucanase activity in leaves > stem > tubers > stolons but not in roots. Increased enzyme activity was recorded for up to 45 days post-treatment.

Field trials for the control of Xiphinema diversicaudatum and Longidorus elongatus in a peat soil, L. macrosoma in a fine sandy loam soil, and L. attenuatus and L. leptocephalus in a sandy soil were done using 1,3-dichloropropene applied at various rates injected to either 15 cm or split injected at cither 15 and 30 cm or 15 and 45 cm. Good control of all the nematode species was obtained at two of the three sites to at least 60 cm though very intensive sampling revealed small residual populations following most treatments. The degree of control achieved at these two sites would probably reduce the risk to high-grade strawberry nursery stock grown on the sites from reservoirs of virus outside the site.

The association of the cell wall degrading enzyme endo-beta-1,4-xylanase (EC 3.2.1.8) with pathogenicity of Mycosphaerella graminicola was examined in planta. The enzyme production of two M. graminicola isolates (T0372 and T0491), as well as their ability to infect seedlings of susceptible wheat cv. Scorpion, was first compared. No significant difference was found between the two isolates regarding spore germination rates, mycelial growth on the leaf surface or direct and stomatal penetrations. However, restricted hyphal growth was observed inside leaves inoculated with T0372, whereas successful mesophyll colonization with a strong intercellular fungal growth was found in leaves infected with T0491. Likewise, T0372 was unable to induce lesions bearing pycnidia and to produce endo-beta-1,4-xylanase activity until 22 days post-inoculation (d.p.i.). On the other hand, significant high increases of both diseased leaf area bearing pycnidia and endo-beta-1,4-xylanase activity were observed between 16 and 22 d.p.i. for T0491 (r = 0·98). The investigation of 24 additional isolates, including the IPO323 and IPO94269 reference isolates, highlighted a strong correlation between endo-beta-1,4-xylanase activity and disease development levels (r = 0·94). This study demonstrates that differences in pathogenicity in M. graminicola are not linked to events on the leaf surface or to frequency of leaf penetration, but to the ability of the fungus to colonize the mesophyll and to produce the cell wall degrading enzyme endo-1,4-beta-xylanase during the necrotrophic phase.

Endo-1,4-beta-glucanases, which can degrade cellulose, have been identified in a number of plant-parasitic nematodes, mainly sedentary endoparasites. This study reports the discovery of four different endoglucanases of glycosyl hydrolase family 5 (GHF5) in the migratory endoparasitic nematode Radopholus similis. Spatial expression of the corresponding genes was analysed by in situ hybridization, which showed the presence of transcripts in the pharyngeal gland cells. A semi-quantitative RT-PCR on different developmental stages was performed to study the temporal expression pattern. Three of the endoglucanase genes showed reduced expression in adult males compared with that in females. This could be explained by the fact that males do not feed and are considered non-parasitic. Only one of the endoglucanase genes was expressed in juveniles. The four corresponding proteins had a putative signal peptide for secretion and a catalytic domain. Two of the proteins had an additional linker and carbohydrate-binding module (CBM). Modelling of the catalytic domain resulted in the α/β-barrel typical for GHF5 endoglucanases. Mapping the conserved amino acids of the four endoglucanases onto the 3D structure revealed that most were positioned near the catalytic centre of the protein, whereas less conserved amino acids occurred more often in the alpha helices, pointing towards the outside of the protein. Analyses of guanine/cytosine (GC) contents and codon adaptation indices indicated that the endoglucanase genes were well adapted to the codon usage of R. similis. The GC and GC3 (GC content of the third position of the codons) contents of the endoglucanases were significantly higher than the average for all Radopholus ESTs (expressed sequence tags).

The inheritance of virulence in Bremia lactucae to match specific resistance factors in lettuce was studied by crossing heterothallic isolates of B. lactucae. Avirulence seemed to be dominant to virulence. Although virulence to some R-factors was inherited at a single locus, thus supporting the hypothesis of a gene-for-gene interaction between B. lactucae and L. sativa, inheritance of virulence to other R-factors was more complex. Two loci seemed to determine virulence to R11; the determinants of virulence to R5, R8 and R10 were either closely linked or allelic; virulence to these loci appeared to be epistatic to virulence to match R9. Virulence to R4 probably involved the interaction of two loci, one of which inhibited avirulence. Determinants of fitness of B. lactucae and possibly genes of minor effect modifying specific virulence factors also segregated. These studies emphasized the potential variability in B. lactucae and implied that isolates should not be assigned to distinct races.

The inheritance of specific virulence in Bremia lactucae was studied by hybridizing two isolates which differed in their virulence on host cultivars carrying the resistance factors R1, R2, R4, R6 and R11. Avirulence on R1, R2, R4 and R11 was dominant to virulence and the segregation conformed to that expected for alleles at single loci, although reactions on R4 were not always dearly defined. The loci conditioning avirulence on R2 and R11 were probably linked. It was difficult to classify the reactions of isolates on R6 and the ratios obtained could not readily be interpreted in Mendelian terms. Since no segregation was observed for reaction on R3, R5, R7, R8, R9 and R10 it was concluded that the genes determining virulence on these factors occurred at the same loci in both isolates, Sexual compatibility type (B1 and B2) segregated with a ratio of 1:1.

SSF-126, (E)-2-methoxyimino-N-methyl-2- (2-phenoxyphenyl)acetamide, inhibited mycelial growth (IC50 7·7 μM) of Pyricularia oryzae. The inhibited mycelium, which was incubated for 30 h in the presence of SSF-126, grew vigorously after transfer to a fungicide-free medium, indicating that SSF-126 is a fungistatic compound. SSF-126 strongly inhibited oxygen consumption by P. oryzae at low concentrations (IC50 0·8 μM). However, the initial inhibition of oxygen consumption was followed by normal mycelial respiration that was insensitive to SSF-126. These results suggest that SSF-126 has a new type of antifungal action.

The occurrence, distribution, and genetic relatedness of double-stranded RNA (dsRNA) components from 36 isolates of Rhizoctonia solani belonging to nine anastomosis groups (AGs) were studied using electrophoretic analysis and RNA–RNA blot hybridization. DsRNA was consistently detected in all 36 isolates. The size of the dsRNA components varied considerably, ranging from 0·74 to 23 kb. Two thirds of isolates possessed different size dsRNA components. Only two of the isolates had small size dsRNA between 0·5 and 1·0 kb. The biotin-labelled dsRNA probes provided the sensitivity and specificity required to study genetic relationships of dsRNA. As little as 10 pg of ‘hybridizing’ dsRNA could be detected using biotin-labelled total dsRNA with no detectable nonspecific-hybridization. Results from several dot-spot as well as RNA–RNA gel hybridization experiments revealed considerable sequence heterogeneity among dsRNA components within each isolate or isolates from the same AG.

Global food production must increase by 50% to meet the projected demand of the world’s population by 2050. Meeting this difficult challenge will be made even harder if climate change melts portions of the Himalayan glaciers to affect 25% of world cereal production in Asia by influencing water availability. Pest and disease management has played its role in doubling food production in the last 40 years, but pathogens still claim 10–16% of the global harvest. We consider the effect of climate change on the many complex biological interactions affecting pests and pathogen impacts and how they might be manipulated to mitigate these effects. Integrated solutions and international co-ordination in their implementation are considered essential. Providing a background on key constraints to food security, this overview uses fusarium head blight as a case study to illustrate key influences of climate change on production and quality of wheat, outlines key links between plant diseases, climate change and food security, and highlights key disease management issues to be addressed in improving food security in a changing climate.

First leaves of barley seedlings grown in solution culture were inoculated with powdery mildew when the second leaf was fully emerged. 14C-labelled carbon dioxide was fed to either first or second leaves of infected and non-infected plants. Translocation of labelled photoassimilates into roots, and partitioning into soluble, storage and structural fractions were studied in tip, mid and basal regions of primary roots 24 h after feeding. Mildew reduced the total activity in the plant, but had little effect on assimilate distribution since the percentage activity translocated to roots was only transiently reduced, approximately 7 days after infection. Reduced import led to a reduction in the specific activity of different fractions within roots, reductions being greatest in root tips. These changes were similar whichever leaf was fed. Changes in specific activity became progressively more pronounced in the 10 days following infection, and were paralleled by a reduction in the mitotic index of root tips. It is concluded that meristematic activity, and thus the growth, of primary roots of barley is particularly sensitive to reductions in photoassimilation caused by powdery mildew infection.

Several published polymerase chain reaction (PCR) primers to identify Pseudomonas syringae pv. actinidiae, the causal organism of bacterial canker of kiwifruit, were found not to be specific. Two new sets of PCR primers, PsaF1/R2 and PsaF3/R4, were designed to be complementary to a portion of the 16S–23S rDNA intertranscribed spacer (ITS) regions. These primers amplified a DNA fragment from strains of P. syringae pv. actinidiae, but not from 56 strains of bacteria from six genera and 17 species, except for a strain of the tea pathogen, P. syringae pv. theae. When tested against DNA extracted from a further 20 strains from Japan, Korea, Italy and the USA deposited in culture collections as P. syringae pv. actinidiae, all except six cultures produced the expected product of 280 bp with PsaF1/R2 and 175 bp with PsaF3/R4. Results of multilocus sequence analysis using five housekeeping genes (gyrB, acnB, rpoD, pgi and cts) showed that none of these six strains was phylogenetically similar to P. syringae pv. actinidiae. In contrast to the P. syringae pv. actinidiae type strain, these strains were positive in the determinative tests for ice nucleation and syringomycin production. It is suggested that these six strains were incorrectly identified as P. syringae pv. actinidiae. It was not possible to distinguish P. syringae pv. actinidiae from the phylogenetically similar P. syringae pv. theae using the ITS, gyrB, acnB, rpoD, pgi or cts gene regions to design PCR primers. Because P. syringae pv. theae is unlikely to be found on kiwifruit, primers PsaF1/R2 and PsaF3/R4 are recommended for screening bacteria isolated from kiwifruit tissue.

A method using consensus PCR followed by oligonucleotide microarray hybridization was developed for identification of phytoplasma 16Sr ribosomal groups. The array consisted of 21– to 33-nt-long oligonucleotides which were designed to hybridize to individual 16Sr groups. Two oligonucleotides were designed to detect all phytoplasma groups. The array could efficiently identify samples from 16SrI, 16SrII, 16SrIII, 16SrV, 16SrVI, 16SrVII, 16SrIX, 16SrX and 16SrXII ribosomal groups. This microarray-based test represents a rapid method for detection of phytoplasmas in unknown samples and for identification of most 16Sr groups.

A new phytoplasma disease of Rehmannia glutinosa var. purpurea was observed in the Czech Republic in 1998. Infected plants showing severely proliferating shoots, leaves reduced in size with vein clearing and chlorosis, shortened internodes and virescent petals died in advanced stages of the disease. Electron microscopy examination of the ultra-thin sections revealed the presence of numerous polymorphic bodies in phloem tissue of leaf midribs and petioles. The disease was successfully transmitted from infected plant via a dodder bridge into periwinkle (Catharanthus roseus). The phytoplasma aetiology of this disease was further confirmed by polymerase chain reaction (PCR) using universal primers R16F2/R16R2. Restriction fragment length polymorphism (RFLP) analysis of amplification products indicated the presence of aster yellows related phytoplasmas (16SrI-B) in naturally infected samples of R. glutinosa var. purpurea and in symptomatic periwinkle after dodder transmission of the agent. A comparison of the amplified sequence with 17 sequences available in the GenBank confirmed the classification of the phytoplasma in the subgroup 16SrI-B. This is the first report of natural occurrence of phytoplasma-associated disease in R. glutinosa var. purpurea.

This study determined the tuf gene sequence of the phytoplasma specific to paulownia witches’-broom from Nanyang, China (hereby designated PaWB-Ny). The PaWB-Ny tuf gene was 1185 nucleotides in length and confirmed that the phytoplasma belongs to subgroup 16SrI-D of aster yellows. Three characteristic GTP-binding protein motifs were identified based on the peptide deduced from the tuf gene sequence. Results suggested that the elongation factor EF-Tu was localized in the cytoplasm and lacked hydrophobic transmembrane domains. Antibodies against PaWB-Ny EF-Tu were prepared by rabbit immunization with glutathione-S-transferase (GST)-tagged EF-Tu fusion protein expressed in Escherichia coli. EF-Tu exhibited a molecular weight of ∼43 kDa and was detected in PaWB-infected paulownia plants by western blot analysis. Indirect enzyme-linked immunosorbent assays (ELISA) and dot blotting analyses were performed with freezing and thawing treatments during antigen preparation. Dilution of extracts to an appropriate scale significantly reduced non-specific reactions. The resultant PaWB EF-Tu antibody reacted with antigens from plants infected with periwinkle virescence and chinaberry tree witches’-broom phytoplasmas, but not those infected with jujube witches’-broom or bishopwood witches’-broom phytoplasma. The EF-Tu was characteristically localized within the phytoplasmal cytoplasm of infected plant phloem tissues.

The results of this study clearly distinguish ApSL from AP phytoplasma, ‘Candidatus Phytoplasma mali’, a member of group 16SrX. Five subgroups of phytoplasma group 16SrI have now been identified in Lithuania: subgroup 16SrI-A in vegetables, ornamentals and oats; subgroup 16SrI-B in ornamentals and woody plants including apple; subgroup16SrI-C in clover and Poa pratensis; and subgroups16SrI-M and 16Sr-L in ornamentals, cereals and woody plants (Staniulis et al., 2000; Jomantiene et al., 2002; Valiunas, 2003). This first report of ‘Candidatus Phytoplasma asteris’ infecting apple in Lithuania has considerable significance for fruit production, as apple is widely cultivated throughout Europe.

Potatoes are the main crop of smallholder farmers in the Valles Cruceños, Santa Cruz Province, Bolivia. During surveys carried out from 2000 to 2003, a disease locally known as ‘brotes grandes’ (BG, ‘big bud’), was prevalent on crops in the valleys of Chilon, Saipina, Pulquina and Comarapa, where up to 90% of plants were affected in some fields. Symptoms included tuber-like growths in leaf axils, varying in size and colour from red to purple or black and bearing terminal, adventitious leaves. Tubers often produced hair-like shoots, reducing their quality and yield. Previously, this syndrome was presumed to be rhizoctoniosis, caused by basal stem infection by Rhizoctonia solani (teleomorph, Thanatephorus cucumeris). However, R. solani was absent from all diseased plants examined in the present study. As other potato diseases similar to BG have been attributed to phytoplasmas in Australia (Harding & Teakle, 1993), Poland (Hamilton, 2000), Canada (Khadhair et al., 2003) and Mexico (Martínez-Soriano et al., 1999), tissues from Bolivian plants were indexed by nested PCR (nPCR) using phytoplasma universal rRNA primer pairs P1/P7 and R16F2n/R16R2. nPCR products resulting from 43/50 BG samples and digested separately with HaeIII, RsaI or AluI endonuclease all produced identical RFLP profiles. RFLP profiles of nPCR products amplified from three samples of the mora-mora vine (Serjania perulacea) with little-leaf symptoms in hedgerows surrounding potato fields in La Tranca, Santa Cruz Province, were indistinguishable from BG profiles. 16S rDNA sequences derived from PCR products shared 99% sequence homology among BG phytoplasmas. Sequences from the phytoplasmas obtained from potato (accession no. AY725209) and from S. perulacea (accession no. AY725210) were each very similar (98%) to that of ash witches’ broom phytoplasma (accession no. AY568302). This is the first report of a phytoplasma of the aster yellows (16SrI) group associated with a potato disease in Bolivia.

A study to investigate the association of phytoplasmas with papaya dieback and citrus decline syndromes in Ethiopia was carried out between July 2009 and February 2010, with sampling performed in major papaya- and citrus-growing areas of the Rift Valley. Samples of plants with symptoms were collected from papaya, citrus and suspected phytoplasma weed hosts and crops in and around the papaya and citrus fields studied. Nested polymerase chain reaction (nested-PCR) was used for initial characterization, using primers that amplify regions of the 16S rRNA and secA genes, and results were then confirmed with rapid real-time group-specific LAMP (loop-mediated isothermal amplification) assays. The results identified the occurrence of a phytoplasma belonging to the stolbur (16SrXII-A) group in papaya plants showing dieback symptoms, whilst no phytoplasmas were found associated with citrus decline. These results contradict previous reports that a 16SrII phytoplasma was associated with both papaya dieback and citrus decline in Ethiopia, but correspond with the association of a 16SrXII phytoplasmas with Nivum-Haamir-Dieback of papaya in Israel and papaya dieback in Australia. No 16SrXII phytoplasmas were found in any of the weeds and potential alternative hosts studied, although 16SrII phytoplasmas were consistently found in Parthenium hysterophorus weed plants. These results indicate that a 16SrXII phytoplasma is associated with papaya dieback in Ethiopia, whilst the causal agent of citrus decline is not a phytoplasma and remains unidentified.

plants growingnearby brinjal (aubergine) fields in Ambedkar Nagar, U.P., India duringMarch 2008. The diseased plants exhibited proliferation of branches withshortened internodes and reduced-size leaves which give rise to the littleleaf appearance. To ascertain the association of a phytoplasma, total DNAwas extracted from ~100 mg of leaf tissue employing a phytoplasmaenrichment procedure (Ahrens & Seemuller, 1992). Direct polymerasechain reaction (PCR) was performed using P1/P6 universal primers specificto 16S rRNA gene of phytoplasmas (Deng & Hiruki, 1991), followed bya nested PCR from a 1:10 dilution of the P1/P6 PCR products usingR16F2n/R16R2 primers (Gundersen & Lee, 1996), which resulted inexpected size bands of ~1·5 kb and ~1·2 kb, respectively, for little leafaffected (2/2) but not symptomless (1/1) samples.Nested PCR amplicons from each sample were sequenced and theconsensus nucleotide sequence deposited in GenBank (Accession No.EU573925). BLAST analysis of the 16S rRNA partial sequence of thephytoplasma identified in little leaf-affected

Peach ( Prunus persica ), family Rosaceae, is native to China . In Canada , the province of Ontario produces 82% of the nation's peach crop. The Canadian Clonal Genebank , also located in Ontario, currently holds 83 accessions of peach. The …

A protocol based on Co-operational PCR has been successfully applied to the detection of phytoplasmas. A triprimer reaction coupled with hybridization using general and specific probes permitted detection of ‘Candidatus Phytoplasma mali’, ‘Ca. Phytoplasma prunorum’ and ‘Ca. Phytoplasma pyri’, and their identification as members of 16S ribosomal quarantine group X. The sensitivity of this method was at least one hundred times greater than conventional PCR and similar to that achieved by nested PCR and real-time PCR. The method was validated by testing field samples collected from Malus, Prunus and Pyrus spp. and Olea europaea and compared with seven phytoplasmas maintained in Catharanthus roseus.

A severe decline of apricot trees, present in Austria for several years, has recently reached notifiable levels. Initial symptoms on affected trees resemble deficiencies in water and nutrient supplies, expressed as leaf rolling, chlorosis and early reddening, leading to sudden dieback during the growing season. Small, wilted fruit and dried leaves may also persist during the autumn. Phytoplasmas belonging to the 16SrX-B subgroup of European stone fruit yellows (ESFY) were identified by nested polymerase chain reaction/restriction fragment length polymorphism analysis in a high percentage of affected trees from two different regions of Austria.