Article

First Report of Leaf Spot on Maize Caused by Curvularia verruculosa in India

Authors:
  • ICAR-National Bureau of Agriculturally Important Microorganisms
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

Curvularia leaf spot affects maize plants worldwide and is commonly caused by Curvularia lunata, C. geniculata, and C. pallescens (Manzar et al. 2022; Manzar et al. 2021; Choudhary et al. 2011). In February 2017, leaf spot symptoms were observed in a Deogaon, (25.74 N, 82.99 E) in Uttar Pradesh, India, with disease incidence of less than 10% of the plants in maize fields. On the leaves and sheaths, variously shaped yellow spots were developed. The spots were 2.5 mm in diameter and frequently grew larger, reaching a diameter of 1 cm. They were encircled by a chlorotic halo with dark borders. The symptomatic tissue showing leaf spots of 10 plants was taken and cut into pieces (4 mm2) then surface sterilized with 1% sodium hypochlorite for 1 min, and rinsed three times with distilled water. The cut leaf tissue was placed on the Petri plate containing potato dextrose agar medium amended with streptomycin sulfate (125 ppm). Then incubated at 25±2°C with a 12-h light and dark period, after 5 days of incubation, five pure cultures were obtained using the hyphal tip technique. The pure culture was incubated at 26±2°C for 10 days. The upper surface of the colony was dark grayish black with fluffy mycelia, and the reverse colony was dark brown. The conidia have three septa, are light brown to dark brown in color, straight to curved, ellipsoidal to fusiform, and have two bigger, darker central cells than terminal cells. On average, conidia are between 27.22 to 31.21 mm long and 10.61 to 12.62 mm wide (n=30). The morphological description is similar to the Curvularia verruculosa morphological traits described by Tandon & Bilgrami (Ellis 1966). Molecular identification was done in addition to supporting morphological identification. The nucleopore GDNA Fungus Kit (Genetix Brand, India) was used to extract the genomic DNA of the E40 isolate. The ITS rDNA region (White et al. 1990) and the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene (Berbee et al. 1999) were amplified through PCR(Manzar et al., 2022).The amplicons were bidirectional sequenced through the Sanger sequencing method. The similarity percentage of E40 isolate matched 100% with MH859788 (CBS444.70 ) of Curvularia verruculosa strain for ITS, and 100% with LT715824 (CBS150.63) of Curvularia verruculosa strain for gpd after Blastn analysis. The gene sequences were deposited to GenBank and accession no. OR262893 for ITS, and LC773704 for gpd were assigned. As a result, C. verruculosa was determined to be the presumed pathogen by both morphology and molecular characteristics. The pathogenicity of E40 isolate was performed twice by spraying (106 conidia/ml in sterile water) onto the leaves of 25 days old maize plant cv. Kanchan (n = 10). Uninoculated healthy maize plants (n=5) were sprayed only with autoclaved water. All pots are kept in a glass house at 25°C±2°C with 90% relative humidity. After 15 days of pathogen inoculation the foliar spots with chlorotic halo, enlarger upto 1cm, and from these spots the identical fungus was reisolated. The reisolated fungus showed similar morphological characteristics to C. verruculosa. Control plants showed no symptoms. C. verruculosa has been previously reported as a causative agent of leaf spot disease in Common beans (Wei et al., 2022), Cotton (Shirsath et al., 2018). To our knowledge, this is the first report of leaf blight caused by C. verruculosa on maize in India.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

Article
Foxtail millet ( Setaria italica ) an economically important crop globally, particularly in China. In 2021, a foxtail millet ear rot disease was first found in Chaoyang City, Liaoning Province, China. Symptomatic tissues were sampled, and the fungal isolate GZWB008 was identified as Curvularia geniculata based on morphological characteristics and ITS, GAPDH and RPB2 gene sequence analyses. Pathogenicity was confirmed by artificial inoculation, which produced symptoms consistent with those observed in the field. Koch's postulates were fulfilled by re‐isolation of C. geniculata from inoculated symptomatic ears. This is the first report confirming C. geniculata as a causal agent of ear rot in foxtail millet, expanding knowledge of its host range and highlighting its potential threat to millet production.
Article
Full-text available
Piriformospora indica is an important endophytic fungus with broad potential for alleviating biotic and abiotic stress on host plants. This study monitored the growth dynamics of P. indica on five commonly used artificial media for microorganisms and analyzed its metabolic characteristics using Biolog Phenotype Microarray (PM) technology. The results showed that P. indica grew fastest on Potato Dextrose Agar (PDA), followed by Kidney Bean Agar (KBA), Alkyl Ester Agar (AEA), Oatmeal Agar (OA), and Luria-Bertani Agar (LB), and the most suitable medium for spore production was OA. Using Biolog PM1-10, 950 metabolic phenotypes of P. indica were obtained. P. indica could metabolize 87.89% of the tested carbon sources, 87.63% of the tested nitrogen sources, 96.61% of the tested phosphorus sources, and 100% of the tested sulfur sources. P. indica displayed 92 kinds of tested biosynthetic pathways, and it could grow under 92 kinds of tested osmotic pressures and 88 kinds of tested pH conditions. PM plates 1-2 revealed 43 efficient carbon sources, including M-Hydroxyphenyl acid, N-Acetyl-D-Glucosamine, Tyramine, Maltotrios, α-D-Glucosine, I-Erythritol, L-Valine, D-Melezitose, D-Tagatose, and Turanose. PM plates 3,6-8 indicated 170 efficient nitrogen sources, including Adenosine, Inosine Allantoin, D, L-Lactamide, Arg-Met, lle-Trp, Ala-Arg, Thr-Arg, Trp-Tyr, Val-Asn, Gly-Gly-D-Leu, Gly-Gly-Phe, and Leu-Leu-Leu. This study demonstrates that P. indica can metabolize a variety of substrates, such as carbon and nitrogen sources, and has a wide range of environmental adaptability. The growth dynamics on artificial culture media and metabolic phenotypes of P. indica can be used to investigate its biological characteristics, screen for more suitable growth and sporulation conditions, and elucidate the physiological mechanisms that enhance the stress resistance of host plants. This study provides a theoretical basis for its better application in agriculture.
Article
Full-text available
Microbial inoculants may be called magical bullets because they are small in size but have a huge impact on plant life and humans. The screening of these beneficial microbes will give us an evergreen technology to manage harmful diseases of cross-kingdom crops. The production of these crops is reducing as a result of multiple biotic factors and among them the bacterial wilt disease triggered by Ralstonia solanacearum is the most important in solanaceous crops. The examination of the diversity of bioinoculants has shown that more microbial species have biocontrol activity against soil-borne pathogens. Reduced crop output, lower yields, and greater cost of cultivation are among the major issues caused by diseases in agriculture around the world. It is universally true that soil-borne disease epidemics pose a greater threat to crops. These necessitate the use of eco-friendly microbial bioinoculants. This review article provides an overview of plant growth-promoting microorganisms bioinoculants, their various characteristics, biochemical and molecular screening insights, and modes of action and interaction. The discussion is concluded with a brief overview of potential future possibilities for the sustainable development of agriculture. This review will be useful for students and researchers to obtain existing knowledge of microbial inoculants, their activities, and their mechanisms, which will facilitate the development of environmentally friendly management strategies for cross-kingdom plant diseases.
Article
Full-text available
Bipolaris species are known to be important plant pathogens that commonly cause leaf spot, root rot, and seedling blight in a wide range of hosts worldwide. In 2017, complex symptomatic cases of maydis leaf blight (caused by Bipolaris maydis) and maize leaf spot (caused by Cur-vularia lunata) have become increasingly significant in the main maize-growing regions of India. A total of 186 samples of maydis leaf blight and 129 maize leaf spot samples were collected, in 2017, from 20 sampling sites in the main maize-growing regions of India to explore the diversity and identity of this pathogenic causal agent. A total of 77 Bipolaris maydis isolates and 74 Curvularia lunata isolates were screened based on morphological and molecular characterization and phylogenetic analysis based on ribosomal markers-nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) region, 28S nuclear ribosomal large subunit rRNA gene (LSU), D1/D2 domain of large-subunit (LSU) ribosomal DNA (rDNA), and protein-coding gene-glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Due to a dearth of molecular data from ex-type cultures, the use of few gene regions for species resolution, and overlapping morphological features, species recognition in Bipolaris has proven difficult. The present study used the multi-gene phylogenetic approach for proper identification and diversity of geographically distributed B. maydis and C. lunata isolates in Indian settings and provides useful insight into and explanation of its quantitative findings .
Article
Common bean is extensively grown and consumed worldwide and it is considered a nutraceutical food and an important source of protein. Leaf spot is one of the most devastating diseases of common bean in tropical and subtropical production areas. In May 2021, this destructive disease was observed in the Guizhou Province of China and resulted in serious economic losses for local agricultural production. Foliar symptoms appeared as small circular tan-colored spots, which then turned dark brown and coalesced into irregular water-soaked necrotic lesions, often leading to leaf wilt or abscission. Fungal isolates were obtained from symptomatic common bean plants and a detailed morphological analysis was performed. Concatenated phylogenetic reconstruction was conducted by amplifying and sequencing the internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (gapdh) and translation elongation factor 1-alpha (tef1-α) genes. Based on morphological characters and phylogenetic analyses, the pathogen was identified as Curvularia verruculosa. In addition, the pathogenicity tests of the isolates reproduced the same symptoms as those observed in the field, and Koch's postulates were confirmed by pathogen re-isolation and identification. To our knowledge, this is the first report of C. verruculosa causing leaf spot on common bean in the world. The current study can provide a foundation for developing effective management strategies, and reducing economic losses in common bean production.
Article
In India, 25.90 million metric tons of maize (Zea mays L.) was produced on 9.63 million hectares, with an average yield of 2.69 metric tons per hectare (Singh and Singh 2018). In March 2017, leaf spot was observed on maize cv. Kanchan during a survey of the Ballia district, with disease severity ranging from 1 to 20% (26.08 N, 83.71 E) in Uttar Pradesh, India. The upper mid canopy of symptomatic plants showed elliptical light brown spots (0.25 to 2.5 mm diameter) surrounded by chlorotic halo lesions with dark margins. Five plants showing leaf spots were collected and the symptomatic tissue was cut into pieces (4 mm 2), surface sterilized with 1% sodium hypochlorite for 1 min, then rinsed three times with distilled water, plated on potato dextrose agar medium amended with streptomycin sulfate (125 ppm), and incubated at 26 C with a 12-h light period. After 5 days of incubation, five pure cultures were obtained using the hyphal tip technique and incubated at 28 C for 15 days. The upper surface of the colony was dark grayish black and the reverse colony was dark brown. Conidiophores on the apex were geniculate, unbranched, and septate. Conidia were spindle to elliptical in shape, light brown, with 3 to 4 septa with an enlarged central cell. Conidial size ranged from 10.0 to 14.1 lm wide and 19.3 to 26.2 lm long (n = 35). Based on morphology, the fungus was tentatively identified as Curvularia geniculata (Tracy & Earle) Boedijn (Hosokawa et al. 2003). Later, genomic DNA was extracted from the E29 isolate using the nucleopore GDNA Fungus Kit (Genetix Brand, India). ITS (White et al. 1990), LSU (Vilgalys and Hester 1990), D1 and D2 region of LSU (O'Donnell 1993), and glyceraldehyde-3-phosphate dehydrogenase (gpd) (Berbee et al. 1999) loci were amplified for molecular identification (Manamgoda et al. 2012). The amplicons were bidi-rectionally sequenced using the Sanger sequencing method. BLASTn analysis of different gene sequences of isolate E29 showed 99 to 100% similarity with sequences of C. geniculata. The gene sequences were submitted to GenBank, and accession numbers were assigned (ITS, MT524330; gpd, LC552684; LSU, MT516309; and D1 and D2 region of LSU, MT533847). The similarity index for ITS, gpd, LSU, and D1 and D2 region of LSU sequences of E29 showed high similarity with isolates of C. geniculata: 99.81% with MH856584 (CBS187.50), 99.52% with KM083609 (CBS187.50), 100% with MH868092 (CBS187.50), and 100% with MH868533 (CBS220.52), respectively. Pathogenicity of the isolate E29 was confirmed by inoculating 25-day-old maize cv. Kanchan (n = 10) with a spore suspension (10 6 spores/ml) prepared from 15-day-old cultures. Using a hand atomizer, the spore suspension was sprayed onto leaves until runoff and the noninoculated plants (n = 10) were sprayed with sterile distilled water. The plants were maintained in a glasshouse at 26 ± 2 C and humidity 90%. After 10 days, elliptical spots with chlorotic halo were observed on inoculated plants similar to those observed in the field, but no symptoms developed on noninoculated plants. C. geniculata was reisolated from symptomatic leaves, and its identity was confirmed by morphological observations. The pathogenicity tests were repeated twice. The disease appears not to be yield limiting; however, it has caused several stakeholder concerns, and anecdotal evidence suggests that maize hybrids may differ in susceptibility. To our knowledge, this is the first report of leaf spot of maize caused by C. geniculata in India, which extends the known agents of maize leaf spot.
Article
We evaluate the phylogenetic distribution of known, highly virulent plant pathogens in the genus Cochliobolus (sexually reproducing species in the Ascomycota, Pleosporaceae) and assess the relationship between Cochliobolus species and species of Curvularia and Bipolaris (asexual states of fungi in the Ascomycota, Pleosporaceae). To infer a phylogeny, we have used two sequence regions: (i) the complete ITS 1, ITS 2, and 5.8S rDNA sequences for 65 fungal isolates and (ii) a 600 bp fragment of the housekeeping gene gpd, coding for glyceraldehyde-3-phosphate dehydrogenase, for 54 isolates. We combined ITS, 5.8S and gpd sequence data from 41 species. In the Cochliobolus clade, 31 out of 32 species fit clearly into one of two groups. One species, Cochliobolus homomorphus, did not fit clearly into either group. The 13 species in Cochliobolus Group 1 grouped together with 100% bootstrap support from the combined ITS/gpd data. This group included Cochliobolus and Bipolaris species that cause serious crop losses, such as Co. sativus, Co. miyabeanus, Co. carbonum and Co. heterostrophus. However, within Group 1, the known, highly virulent pathogens did not form a monophyletic group of species. Average substitution levels between pairs of species in the Group 1 were low, about 1.7% in the ITS region, suggesting that these species had radiated rapidly and recently. The 18 species in Cochliobolus Group 2 formed a monophyletic group in 96% of parsimony bootstrap replicates of the combined ITS and gpd data. The Cochliobolus species that were transferred into the segregate genus Pseudocochliobolus were in this second group. This study included 9 Curvularia and Bipolaris species without known sexual states and they all appear to be recently derived from among sexual species of Cochliobolus. Both Curvularia and Bipolaris were polyphyletic, but only Bipolaris states were associated with Group 1 Cochliobolus species. Both Curvularia and Bipolaris states were associated with species in Cochliobolus Group 2.
Article
The teleomorph of Curvularia verruculosa, the new species Pseudocochliobolus verruculosus, was produced in culture by crossing compatible isolates. The species is heterothallic, and two mating types were detected in conidium isolates both from Burma and Japan. Conidium morphology is variable among isolates, and varies within an isolate on different media.
Article
The finding described in this study is the first report of leaf spot disease of cotton caused by Curvularia verruculosa surveyed in the state of Maharashtra (India). The isolated phytopathogenic fungal strain was identified using morphological characteristics and molecular identification of ITS gene sequence (MF784436) and D1D2 region of LSU gene (KY978073). The ability of fungal strain to secrete hydrolytic enzymes viz., pectinase, xylanase, protease, cellulase and lipase was detected. The secretion profile of hydrolytic enzymes by C. verruculosa was also examined in planta and in vitro. The secretion of cellulase, xylanase and protease was found to be inducible on cotton-stalk powder containing media; while secretion of pectinase and lipase was constitutive in glucose containing medium. The hydrolytic enzymes secretion during etiological progression of disease was detected on cotton leaves at regular interval of 24 h up to 10 days. A significant correlation (P < 0.05) was observed between hydrolytic enzymes secretion and disease severity index. The increased level of hydrolytic enzymes in infected plant sample indicates their role in disease progression. The newly documented fungal phytopathogen Curvularia verruculosa was deposited at National Fungal Culture Collection of India, Pune with accession number of NFCCI-4119.