Unraveling the dynamics of wheat leaf blight complex: isolation, characterization, and insights into pathogen population under Indian conditions

Wheat, a staple food crop for 35% of the global population, faces a threat from Helminthosporium leaf blight (HLB), a complex of spot blotch (Bipolaris sorokiniana) and tan spot (Pyrenophora-tritici-repentis) diseases under warm and humid conditions. However, in Indian conditions, the knowledge of existing pathogen populations associated with the HLB complex is limited and largely dominated by only B. sorokiniana (spot blotch). To address this, diseased samples were collected from all six wheat growing zones during 2020–2022. The pathogenic species were identified through in-depth morphological characterization, supplemented with ITS-rDNA and GAPDH sequence analysis, a diagnostic SCAR marker, and pathogenicity studies on two wheat varieties: Sonalika and HD2733. The 32 isolates collected from 10 different states consist of B. spicifera (12.5% of all isolates), Exserohilum rostratum (9.3%), Bipolaris oryzae (3.1%), and B. sorokiniana (75%). B. sorokiniana exhibited the highest disease severity on both varieties. Other lesser-known pathogenic species also produced comparable disease severity as B. sorokiniana isolates and, therefore are economically important. Unraveling pathogen composition and biology aids in disease control and resistance breeding. Our study highlights economically impactful and lesser-known pathogenic species causing wheat leaf blight/spot blotch in India, guiding both current management and future resistance breeding strategies in plant pathology

Differential gene expression analysis in germinating and dormant teliospores of Tilletia indica using RNA seq approach

Karnal bunt of wheat is an important quarantine disease that interrupts India’s wheat trade in the international market. The whole transcriptome of germinating and dormant teliospores of Tilletia indica was performed using the RNA Seq approach to identify germination-related genes. Approximately 63 million reads were generated using the RNA sequencing by the Illumina NextSeq500 platform. The high-quality reads were deposited in NCBI SRA database (accession: PRJNA522347). The unigenes from the pooled teliospores were 16,575 having unigenes length of 28,998,753 bases. The high-quality reads of germinating teliospores mapped on to 21,505 predicted CDSs. 9,680 CDSs were common between dormant and germinating teliospores of T. indica. 11,825 CDSs were found to be in germinating teliospores while only 91 were unique in dormant spores of pathogen. The pathway analysis showed the highest number of pathways was found in germinating spores than dormant spores. The highest numbers of CDSs were found to be associated with translation (431 in number), transport and catabolism (340), signal transduction (326), and carbohydrate metabolism (283). The differential expression analysis (DESeq) of germinating and dormant teliospores showed that 686 CDS were up-regulated and 114 CDS were down-regulated in the germinating teliospores. Significant germination-related genes in the spores were validated using qPCR analysis. Ten genes viz. Ti3931, Ti6828, Ti7098, Ti7462, Ti7522, Ti 9289, Ti 8670, Ti 7959, Ti 7809,and Ti10095 were highly up-regulated in germinated teliospores which may have role in germination of spores.Further, these differentially expressed genes provide insights into the molecular events. This first study of transcriptome will be helpful to devise better management strategies to manage Karnal bunt disease

Transcriptome analysis of Bipolaris sorokiniana - Hordeum vulgare provides insights into mechanisms of host-pathogen interaction

Spot blotch disease incited by Bipolaris sorokiniana severely affects the cultivation of barley. The resistance to B. sorokiniana is quantitative in nature and its interaction with the host is highly complex which necessitates in-depth molecular analysis. Thus, the study aimed to conduct the transcriptome analysis to decipher the mechanisms and pathways involved in interactions between barley and B. sorokiniana in both the resistant (EC0328964) and susceptible (EC0578292) genotypes using the RNA Seq approach. In the resistant genotype, 6,283 genes of Hordeum vulgare were differentially expressed out of which 5,567 genes were upregulated and 716 genes were downregulated. 1,158 genes of Hordeum vulgare were differentially expressed in the susceptible genotype, out of which 654 genes were upregulated and 504 genes were downregulated. Several defense-related genes like resistant gene analogs (RGAs), disease resistance protein RPM1, pathogenesis-related protein PRB1-2-like, pathogenesis-related protein 1, thaumatin-like protein PWIR2 and defensin Tm-AMP-D1.2 were highly expressed exclusively in resistant genotype only. The pathways involved in the metabolism and biosynthesis of secondary metabolites were the most prominently represented pathways in both the resistant and susceptible genotypes. However, pathways involved in MAPK signaling, plant-pathogen interaction, and plant hormone signal transduction were highly enriched in resistant genotype. Further, a higher number of pathogenicity genes of B. sorokiniana was found in response to the susceptible genotype. The pathways encoding for metabolism, biosynthesis of secondary metabolites, ABC transporters, and ubiquitin-mediated proteolysis were highly expressed in susceptible genotype in response to the pathogen. 14 and 11 genes of B. sorokiniana were identified as candidate effectors from susceptible and resistant host backgrounds, respectively. This investigation will offer valuable insights in unraveling the complex mechanisms involved in barley- B. sorokiniana interaction

Assessment of fungal and bacterial bioagents against Bipolaris sorokiniana inciting spot blotch on barley (Hordeum vulgare)

In India, spot blotch disease caused by Bipolaris sorokiniana is one of the major constraints in barley (Hordeum vulgare L.) production. The present study was carried out during 2022–23 at ICAR-Indian Agriculture Research Institute, New Delhi to identify promising biocontrol agents, which can act as eco-friendly alternatives to chemicals against Bipolaris sorokiniana inciting spot blotch on barley (Hordeum vulgare L.). In this study, 18 isolates of Trichoderma asperellum, 15 isolates of Trichoderma harzianum and 5 bacterial species were tested against B. sorokiniana under in vitro and in planta conditions. In the dual culture assays, Trichoderma asperellum 8686 and Trichoderma asperellum 8687 showed significantly highest per cent disease inhibition of 71.73% and 71.37% respectively. Among bacterial strains, Pseudomonas fluorescens and Bacillus amyloliquefaciens showed significantly good per cent disease inhibition of 64.09% and 57.09% respectively. B. subtilis and Pantoea spp. did not show any pathogen inhibition. In addition, the superior bioagents were also screened out. In the studies on in planta assays, Bacillus amyloliquefaciens (per cent disease control-55.19%) was found most effective for seed treatment against B. sorokiniana. Post-inoculation with biocontrol agents revealed that Bacillus amyloliquefaciens was at par with Trichoderma asperellum and Pseudomonas fluorescens against B. sorokiniana. Additionally, a combination of seed treatment, pre and post-inoculation treatment of biocontrol agents revealed that Bacillus amyloliquefaciens and Trichoderma asperellum 8686 were at par with Trichoderma harzianum (Pusa Th3) against B. sorokiniana. Overall, Bacillus amyloliquefaciens was more effective and consistent to manage spot blotch disease

First report of Leaf Spot Disease Caused by Exserohilum rostratum on Bottle Gourd in India

Not AvailableBottle gourd (Lagenaria siceraria) is a medicinal and nutritious domesticated cucurbit vegetable having annual production of 2.37 million metric tons with a pantropical distribution in India. In August 2017, typical symptoms of leaf spot disease were observed on 3-month-old bottle gourd leaves sampled from open fields in Karnal, India, of sporadic level of about 10% severity. During monsoon season, initial symptoms appeared mostly on young leaves with grayish white to brown lesions surrounded by a yellowish halo, measuring 0.5 to 1.7 × 0.4 to 0.8 cm. These lesions expanded gradually from circular to cylindrical, coalescing and covering almost the entire leaf and ultimately leading to blighting of foliage and defoliation. Symptomatic advancing lesions leaf bits of 4 to 6 mm were surface sterilized with 0.1% HgCl2 for 45 s, rinsed three times in sterile distilled water, placed onto potato dextrose agar (PDA) medium, and incubated at 27°C for 7 to 10 days. The emerging fungal colonies on plates were circular, gray to brown, with abundant cottony aerial mycelium. Conidia were straight to slightly curved, ellipsoidal to narrowly obclavate or rostrate, brown, 5 to 14 septate, with basal septum darker and thicker than other septa, 22.4 to 154.4 × 8 to 14.7 μm, with a distinctly protruding basal hilum. Conidiophores were single, cylindrical, dark brown, geniculate, and 4 to 6 μm thick with a swollen basal cell. Further, it was identified by Indian Type Culture Collection, New Delhi (no. 10.643.17) as E. rostratum. For molecular identification, genomic DNA of isolate Ex-1 was extracted from a single conidial culture, and the internal transcribed spacer regions of rDNA (ITS1-5.8S-ITS4) were amplified and sequenced with universal primers ITS1 and ITS4 (Lin et al. 2011) and deposited in GenBank (MG711857). BLASTn analysis revealed 100% identity with E. rostratum isolates (GenBank accession nos. JN711432.1, FJ949084.1, and KU204880.1). A partial sequence of glyceraldehyde-3-phosphate dehydrogenase-like (Gpd) gene of Ex-1 was also amplified using primers GPD1 and GPD2 (Berbee et al. 1999; Liu et al. 2016), sequenced, and deposited in GenBank (MG918126). BLASTn analysis revealed 99% sequence similarity with an E. rostratum isolate (KU935741.1). Based on morphological characteristics and molecular analysis, the isolate was identified as E. rostratum (Drechs.) Leonard & Suggs (Ahmadpour et al. 2013; Lin et al. 2011). A pathogenicity experiment was conducted using five bottle gourd plants (three leaves inoculated in each plant). Newly matured leaves were inoculated by double spray at a 24-h interval with isolate Ex-1 conidial suspension (105 conidia/ml). Leaves sprayed with sterile water served as a negative control. Right after inoculation, plants were covered with plastic bags to maintain high relative humidity and incubated at ≈30°C with a natural photoperiod. Seven days later, all pathogen-inoculated plants showed symptoms identical to those observed in the field, whereas control plants remained asymptomatic. Koch’s postulates were proved with the reisolation of the fungus from leaves. E. rostratum has been reported as the causal agent of leaf spots on Zea mays, Ananas comosus, Musa paradisiaca, and Hevea brasiliensis in China (Liu et al. 2016). To our knowledge, this is the first report of E. rostratum causing leaf spot disease in bottle gourd in India. This disease occurred in quite a large bottle gourd area, and identification of pathogens was essential for developing integrated pest management strategies.Not Availabl

Differential gene expression analysis in germinating and dormant teliospores of Tilletia indica using RNA seq approach

214-223Karnal bunt of wheat is an important quarantine disease that interrupts India’s wheat trade in the international market. The whole transcriptome of germinating and dormant teliospores of Tilletia indica was performed using the RNA Seq approach to identify germination-related genes. Approximately 63 million reads were generated using the RNA sequencing by the Illumina NextSeq500 platform. The high-quality reads were deposited in NCBI SRA database (accession: PRJNA522347). The unigenes from the pooled teliospores were 16,575 having unigenes length of 28,998,753 bases. The high-quality reads of germinating teliospores mapped on to 21,505 predicted CDSs. 9,680 CDSs were common between dormant and germinating teliospores of T. indica. 11,825 CDSs were found to be in germinating teliospores while only 91 were unique in dormant spores of pathogen. The pathway analysis showed the highest number of pathways was found in germinating spores than dormant spores. The highest numbers of CDSs were found to be associated with translation (431 in number), transport and catabolism (340), signal transduction (326), and carbohydrate metabolism (283). The differential expression analysis (DESeq) of germinating and dormant teliospores showed that 686 CDS were up-regulated and 114 CDS were down-regulated in the germinating teliospores. Significant germination-related genes in the spores were validated using qPCR analysis. Ten genes viz. Ti3931, Ti6828, Ti7098, Ti7462, Ti7522, Ti 9289, Ti 8670, Ti 7959, Ti 7809,and Ti10095 were highly up-regulated in germinated teliospores which may have role in germination of spores.Further, these differentially expressed genes provide insights into the molecular events. This first study of transcriptome will be helpful to devise better management strategies to manage Karnal bunt disease

Multilocus Sequence Typing and Single Nucleotide Polymorphism Analysis in <i>Tilletia indica</i> Isolates Inciting Karnal Bunt of Wheat

Karnal bunt of wheat is an internationally quarantined disease affecting trade, quality, and production of wheat. During 2015–2016, a severe outbreak of Karnal bunt disease occurred in north-western plain zone of India. The present study was undertaken to decipher genetic variations in Indian isolates of Tilletia indica collected from different locations. Seven multilocus sequence fragments were selected to differentiate and characterize these T. indica isolates. A phylogenetic tree constructed based on pooled sequences of actin-related protein 2 (ARP2), β-tubulin (TUB), eukaryotic translation initiation factor 3 subunit A (EIF3A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone 2B (H2B), phosphoglycerate kinase (PGK), and serine/threonine-protein kinase (STPK) showed that isolate KB-11 (Kaithal, Haryana) was highly conserved as it was located in cluster 1 and has the maximum sequence similarity with the reference strain. Other isolates in cluster 1 included KB-16 and KB-17, both from Uttar Pradesh, and KB-19 from Haryana. Isolates KB-07 (Jind, Haryana) and KB-18 (Mujaffar Nagar, Uttar Pradesh) were the most diverse and grouped in a subgroup of cluster 2. Maximum numbers of single nucleotide polymorphisms (SNPs) (675) were in the PGK gene across the T. indica isolates. The minimum numbers of SNPs (67) were in KB-11 (Kaithal, Haryana), while the maximum number of SNPs (165) was identified in KB-18, followed by 164 SNPs in KB-14. KB-18 isolate was found to be the most diverse amongst all T. indica isolates. This first study on multilocus sequence typing (MLST) revealed that the population of T. indica was highly diverse

Development of real time PCR assay for detection and quantification of teliospores of <i>Tilletia indica </i> in soil

549-554Tilletia indica, commonly called Karnal bunt, is an internationally quarantined wheat fungal pathogen which affects commercial seed trading as well as the quality of wheat grain for consumption. The teliospores of Tilletia indica surviving in soil serve as the primary source of inoculum and play a major role in disease development. Proper identification and detection of T. indica teliospores based on morphological features and germination of teliospores is time consuming and tedious. In this study, we validated PCR based species-specific primer which amplified 570 bp fragments using ITSKB primers. Further, the real time PCR assay was developed and standardized for detection and quantification of teliospores in soil. The (R2) correlation coefficient (0.994) between CT values and DNA concentrations showed the accuracy of qPCR based quantification. The sensitivity of qPCR marker was 100 fg. Thirteen field soil samples were assessed by qPCR for quantification of teliospore DNA. Low fungal DNA (15135.61 fg) was detected in field soil (K10) from Karnal, Haryana, India while high DNA concentration (3.31 ng) was detected in field soil from IARI, New Delhi (K4). The qPCR assay was done to correlate DNA concentration and number of teliospores per gram soil. The 125.89 fg DNA concentration of T. indica detected corresponding limit of 14 teliospores. Minimum detection limit in terms of teliospores count was 14. The teliospores recovered from Karnal and IARI farm soils by centrifugation method were 450 and 1341, respectively while the qPCR assay based analysis detected higher number of teliospores ranging 1762 to 368332 teliospores. Thus, the developed qPCR diagnostic marker could be used for accurate, reliable and rapid detection of teliospores in soil which would further help in monitoring, quantifying teliosporic load and threshold level of inoculum in soil

Data_Sheet_1_Unraveling the dynamics of wheat leaf blight complex: isolation, characterization, and insights into pathogen population under Indian conditions.docx

Wheat, a staple food crop for 35% of the global population, faces a threat from Helminthosporium leaf blight (HLB), a complex of spot blotch (Bipolaris sorokiniana) and tan spot (Pyrenophora-tritici-repentis) diseases under warm and humid conditions. However, in Indian conditions, the knowledge of existing pathogen populations associated with the HLB complex is limited and largely dominated by only B. sorokiniana (spot blotch). To address this, diseased samples were collected from all six wheat growing zones during 2020–2022. The pathogenic species were identified through in-depth morphological characterization, supplemented with ITS-rDNA and GAPDH sequence analysis, a diagnostic SCAR marker, and pathogenicity studies on two wheat varieties: Sonalika and HD2733. The 32 isolates collected from 10 different states consist of B. spicifera (12.5% of all isolates), Exserohilum rostratum (9.3%), Bipolaris oryzae (3.1%), and B. sorokiniana (75%). B. sorokiniana exhibited the highest disease severity on both varieties. Other lesser-known pathogenic species also produced comparable disease severity as B. sorokiniana isolates and, therefore are economically important. Unraveling pathogen composition and biology aids in disease control and resistance breeding. Our study highlights economically impactful and lesser-known pathogenic species causing wheat leaf blight/spot blotch in India, guiding both current management and future resistance breeding strategies in plant pathology.</p