Characterization of field isolates of Fusarium spp. from eggplant in India for species complexity and virulence.

Eggplant wilt, despite emerging as a severe disease in India, the etiology must be better studied for its species' complexity and variability. The identity of fungal isolates associated with eggplants of India was established morphologically followed by sequencing and phylogenetic analysis. Three species, Fusarium falciforme, Fusarium incarnatum and Fusarium proliferatum, were observed for the first time in India. The isolates were tested for pathogenicity. Though all of them were pathogenic, the isolates displayed varying degrees of virulence. In further studies, the genetic relatedness of the isolates for virulence was assessed with candidate avirulent (SIX effectors), virulent (Fow1 and Fow2) and SSR markers. The SIX effector genes could not delineate the virulent isolates and were expressed in some non-F. oxysporum isolates for the first time. Likewise, the virulent genes, Fow1 for expression across the isolates and Fow2 for random expression across the isolates, were unsuitable markers for identifying the virulent groups. Hence, the F. oxysporum and F. solani isolates were genotyped with SSR markers. Though the clustering did not correlate with their virulence levels, the dendrogram grouping revealed variability among the F. oxysporum and F. solani isolates. This study concludes that although multiple species of Fusarium are associated with eggplant wilt in India, only F. oxysporum and F. solani are widespread in the surveyed areas. Though the three markers could not delineate the race specificity of the isolates, only the SSR makers could identify the genetic variability and hence, would help screen eggplant germplasm for fusarium wilt resistance.

Expression analysis of genes involved in teliospores germination of Tilletia indica inciting Karnal bunt of wheat.

BACKGROUND: Karnal bunt of wheat is an important quarantine disease, incited by Tilletia indica. It limits India's trade in wheat export. The teliospores are major source of inoculum to initiate and spread the Karnal bunt disease. The study aimed to identify the germination-related genes in the teliospores of T. indica. METHODS AND RESULTS: The candidate genes in the teliospores germination were identified through the differential gene expression analysis with suitable bioinformatics analysis. Keeping in soil-borne nature of fungi, the teliospores of T. indica (2015 and 2018) were subjected to the qPCR analysis. 20 candidate genes were identified having role in germination of teliospores of T. indica. Twenty genes, viz. Ti9297 (9.31, 7.87-fold), Ti8696 (5.13, 6.54-fold), Ti7699 (8.9, 7.7-fold), Ti7858 (10.33, 6.21-fold), Ti7954 (7.46, 5.54-fold), Ti7739 (5.46, 6.46-fold), Ti9665 (10.74, 7.64-fold), Ti9335 (6.75, 4.36-fold), Ti8396 (9.35, 7.72-fold), Ti8126 (8.87, 11.31-fold), Ti7326 (6.04, 7.7-fold), Ti10208 (13.83, 5.81-fold), Ti12356 (7.83, 8.02-fold), Ti14271 (9.98, 6.32-fold), Ti9234 (11.2, 8.72-fold), Ti 8876 (6.47, 3.55-fold), Ti 10,606 (4.97, 2.35-fold), Ti7758 (10.33, 8.78-fold), Ti4692 (6.89, 9.88-fold), and Ti3932 (5.77, 4.5-fold) were found highly expressed in the germinating teliospores of 2015 and 2018, respectively. Eight genes (Ti508, Ti4152, Ti5346, Ti2375, Ti3739, Ti1134, Ti4399, and Ti4422) were downregulated in the germinating teliospores but these eight genes were showed higher expression in the dormant teliospores. CONCLUSIONS: Twenty candidate genes were upregulated in the germinating teliospores are supposed to be involved in the process of germination. Eight genes were downregulated which were related to the process of the dormancy of teliospores. The study will be helpful to devise the newer management strategies for Karnal bunt disease of wheat.

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

[This corrects the article DOI: 10.3389/fmicb.2024.1287721.].

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.

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.

Chemo-profiling of Purpureocillium lilacinum and Paecilomyces variotii isolates using GC-MS analysis, and evaluation of their metabolites against M. incognita.

Nematophagous fungi are the best alternatives to chemical nematicides for managing nematodes considering environmental health. In the current study, activity of metabolites from ten isolates of Purpureocillium lilacinum (Thom) Luangsa-ard (Hypocreales: Ophiocordycipitaceae) and two isolates of Paecilomyces variotii Bainier (Eurotiales: Trichocomaceae), were examined to inhibit the hatching of Meloidogyne incognita (Kofoid & White) Chitwood (Tylenchida: Heteroderidae) eggs. At 100%, 50%, and 25% concentrations, respectively, the culture filtrate of the isolate P. lilacinum 6887 prevented 97.55%, 90.52%, and 62.97% of egg hatching. Out of all the isolates, Pl 6887, Pl 6553, and Pl 2362 showed the greatest results in the hatching inhibition experiment.Gas chromatography-mass spectrometry (GC-MS) analysis revealed a variety of nematicidal compounds from different isolates. A total of seven nematicidal compounds, including four very potent nematicidal fatty acids were found in the isolate Pl 6553. Secondary metabolites of the same isolate possess the highest M. incognita juvenile mortality, i.e., 43.33% and 92% after 48 hrs of treatment at 100 and 200 ppm concentrations, respectively. Significant difference was observed in juvenile mortality percentage among the isolate having highest and lowest nematicidal compounds. Nematicidal fatty acids like myristic and lauric acid were found for the first time in P. lilacinum. Multiple vacuole-like droplets were found inside the unhatched eggs inoculated with the culture filtrate of isolate Pl 6887, and also in the juveniles that perished in the ethyl acetate extract of isolate Pl 6553.

Draft genome sequencing of Tilletia caries inciting common bunt of wheat provides pathogenicity-related genes.

Common bunt of wheat caused by Tilletia caries is an important disease worldwide. The T. caries TC1_MSG genome was sequenced using the Illumina HiSeq 2500 and Nanopore ONT platforms. The Nanopore library was prepared using the ligation sequencing kit SQK-LSK110 to generate approximately 24 GB for sequencing. The assembly size of 38.18 Mb was generated with a GC content of 56.10%. The whole genome shotgun project was deposited at DDBJ/ENA/GenBank under the accession number JALUTQ000000000. Forty-six contigs were obtained with N50 of 1,798,756 bp. In total, 10,698 genes were predicted in the assembled genome. Out of 10,698 genes, 10,255 genes were predicted significantly in the genome. The repeat sequences made up approximately 1.57% of the genome. Molecular function, cellular components, and biological processes for predicted genes were mapped into the genome. In addition, repeat elements in the genome were assessed. In all, 0.89% of retroelements were observed, followed by long terminal repeat elements (0.86%) in the genome. In simple sequence repeat (SSR) analysis, 8,582 SSRs were found in the genome assembly. The trinucleotide SSR type (3,703) was the most abundant. Few putative secretory signal peptides and pathogenicity-related genes were predicted. The genomic information of T. caries will be valuable in understanding the pathogenesis mechanism as well as developing new methods for the management of the common bunt disease of wheat.

Transcriptome Analysis of Wheat-Tilletia indica Interaction Provides Defense and Pathogenesis-Related Genes.

Karnal bunt (Tilletia indica Mitra) is an internationally quarantined disease of wheat. Until now, very little information has been available on the molecular basis of resistance and pathogenicity of T. indica. To investigate the molecular basis of host−pathogen interaction, the transcriptome of T. indica inoculated resistant (HD29) and susceptible (WH542) genotypes of wheat were analyzed. Approximately 58 million reads were generated using RNA sequencing by the Illumina NextSeq500 platform. These sequence reads were aligned to a reference genome of wheat to compare the expression level of genes in resistant and susceptible genotypes. The high-quality reads were deposited in the NCBI SRA database (SRP159223). More than 80,000 genes were expressed in both the resistant and susceptible wheat genotypes. Of these, 76,088 were commonly expressed genes, including 3184 significantly upregulated and 1778 downregulated genes. Four thousand one hundred thirteen and 5604 genes were exclusively expressed in susceptible and resistant genotypes, respectively. Based on the significance, 503 genes were upregulated and 387 genes were downregulated. Using gene ontology, the majority of coding sequences were associated with response to stimuli, stress, carbohydrate metabolism, developmental process, and catalytic activity. Highly differentially expressed genes (integral component of membrane, exonuclease activity, nucleic acid binding, DNA binding, metal ion binding) were validated in resistant and susceptible genotypes using qPCR analysis and similar expression levels were found in RNA-Seq. Apart from the wheat, the mapping of T. indica was 7.07% and 7.63% of resistant and susceptible hosts, respectively, upon infection, which revealed significant pathogenesis-related genes. This first study provided in-depth information and new insights into wheat−T. indica interaction for managing Karnal bunt disease of wheat.

Whole-genome sequence analysis of Bipolaris sorokiniana infecting wheat in India and characterization of ToxA gene in different isolates as pathogenicity determinants.

Spot blotch disease of wheat caused by Bipolaris sorokiniana Boerma (Sacc.) is an emerging problem in South Asian countries. Whole genome of a highly virulent isolate of B. sorokiniana BS112 (BHU, Uttar Pradesh; Accession no. GCA_004329375.1) was sequenced using a hybrid assembly approach. Secreted proteins, virulence gene(s), pathogenicity-related gene(s) were identified and the role of ToxA gene present in this genome, in the development of disease was recognized. ToxA gene (535 bp) was analyzed and identified in the genome of B. sorokiniana (BS112) which revealed 100% homology with the ToxA gene of Pyrenophora tritici repentis (Accession no. MH017419). Furthermore, ToxA gene was amplified, sequenced and validated in 39 isolates of B. sorokiniana which confirmed the presence of ToxA gene in all the isolates taken for this study. All ToxA sequences were submitted in NCBI database (MN601358-MN601396). As ToxA gene interacts with Tsn1 gene of host, 13 wheat genotypes were evaluated out of which 5 genotypes (38.4%) were found to be Tsn1 positive with more severe necrotic lesions compared to Tsn1-negative wheat genotypes. In vitro expression analysis of ToxA gene in the pathogen B. sorokiniana using qPCR revealed maximum upregulation (14.67 fold) at 1st day after inoculation (DAI) in the medium. Furthermore, in planta expression analysis of ToxA gene in Tsn1-positive and Tsn1-negative genotypes, revealed maximum expression (7.89-fold) in Tsn1-positive genotype, Agra local at 5th DAI compared to Tsn1-negative genotype Chiriya 7 showing minimum expression (0.048-fold) at 5th DAI. In planta ToxA-Tsn1 interaction studies suggested that spot blotch disease is more severe in Tsn1-positive genotypes, which will be helpful in better understanding and management of spot blotch disease of wheat. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03213-3.

Genome-Wide Association Mapping of Virulence Genes in Wheat Karnal Bunt Fungus Tilletia indica Using Double Digest Restriction-Site Associated DNA-Genotyping by Sequencing Approach.

Tilletia indica is a quarantine fungal pathogen that poses a serious biosecurity threat to wheat-exporting countries. Acquiring genetic data for the pathogenicity characters of T. indica is still a challenge for wheat breeders and geneticists. In the current study, double digest restriction-site associated-DNA genotyping by sequencing was carried out for 39 T. indica isolates collected from different locations in India. The generated libraries upon sequencing were with 3,346,759 raw reads on average, and 151 x 2 nucleotides read length. The obtained bases per read ranged from 87 Mb in Ti 25 to 1,708 Mb in Ti 39, with 505 Mb on average per read. Trait association mapping was performed using 41,473 SNPs, infection phenotyping data, population structure, and Kinship matrix, to find single nucleotide polymorphisms (SNPs) linked to virulence genes. Population structure analysis divided the T. indica population in India into three subpopulations with genetic mixing in each subpopulation. However, the division was not in accordance with the degree of virulence. Trait association mapping revealed the presence of 13 SNPs associated with virulence. Using sequences analysis tools, one gene (g4132) near a significant SNP was predicted to be an effector, and its relative expression was assessed and found upregulated upon infection.

Multilocus Sequence Typing and Single Nucleotide Polymorphism Analysis in Tilletia indica 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), ß-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.

First Draft Genome Sequence of Wheat Spot Blotch Pathogen Bipolaris sorokiniana BS_112 from India, Obtained Using Hybrid Assembly.

Bipolaris sorokiniana is a devastating fungal pathogen causing spot blotch of wheat. We report here the first draft genome of Bipolaris sorokiniana strain BS_112 from India using sequence reads from the Ion Torrent, Illumina HiSeq, and Nanopore platforms. The genome size was estimated at 35.64 Mb with an average G+C content of 50.20%.

Mapping of stripe rust resistance QTL in Cappelle-Desprez × PBW343 RIL population effective in northern wheat belt of India.

Stripe rust caused by Puccinia striiformis f. sp. tritici is most important and devastating disease of wheat worldwide, which affects the grain yields, quality and nutrition. To elucidate, the genetic basis of resistance, a mapping population of recombinant inbred lines was developed from a cross between resistant Cappelle-Desprez and susceptible cultivar PBW343 using single-seed descent. Variety PBW343 had been one of the most popular cultivars of North Western Plains Zone, for more than a decade, before succumbing to the stripe rust. Cappelle-Desprez, a source of durable adult plant resistance, has maintained its resistance against stripe rust for a long time in Europe. Map construction and QTL analysis were completed with 1012 polymorphic (DArT and SSR) markers. Screenings for stripe rust disease were carried out in field condition for two consecutive crop seasons (2012-2013 and 2013-2014). Susceptible parent (PBW343) achieved a significant level of disease i.e., 100 % in both the years. In present investigations, resistance in Cappelle-Desprez was found stable and response to the rust ranged from 0 to 1.5 % over the years. The estimated broad-sense heritability (h 2) of stripe rust rAUDPC in the mapping population was 0.82. The relative area under the disease progress curve data showed continuous distributions, indicating that trait was controlled multigenically. Genomic region identified on chromosome 2D, was located within the short arm, with flanking markers (Xgwm484-Xcfd73), explained phenotypic variation (PVE) ranged from 13.9 to 31.8 %. The genomic region identified on chromosome 5B was found with the effect of maximum contribution with flanking DArT markers (1376633|F|0-1207571|F|0), PVE ranged from 24 to 27.0 %. This can, therefore, be utilized for marker assisted selection in developing much needed stripe rust resistant lines for the northern wheat belt of India.