First report of a 'Candidatus phytoplasma asteris' related strain (16SrI) associated with phyllody and witches'-broom symptom in Pisum sativum in Hisar, India.

Pea (Pisum sativum L.) is a leguminous vegetable crop, and India holds the fourth position in the production, primarily contributed by three major states: Uttar Pradesh, Madhya Pradesh, and Punjab (Anonymous, 2022). However, a survey conducted in February 2023 at the National Seed Corporation Farm (15 hectares) in Hisar, Haryana, revealed deformities in the growth of some pea plants. Approximately 10% of these plants exhibited a distinct bushy appearance, accompanied by phyllody and witches'-broom symptoms, characterized by deformed leaves and short internodes (Fig. 1). In response to these observed anomalies, a detailed molecular analysis was conducted at the Plant Pathology Laboratory, IARI, New Delhi. The investigation involved the collection of ten samples each from symptomatic and asymptomatic plants, and DNA was extracted from 100 mg leaf midribs using the CTAB method (Ahrens and Seemüller, 1992). The extracted DNA (100 ng/µl) along with one positive (Catharanthus roseus from glass house, 16SrII-D group) and one negative (without template DNA), served as a template for PCR reactions targeting the 16Sr RNA and secA genes. Universal primers P1/P7 and secAfor1/secArev3 were employed in the first round of PCR for the respective genes. Subsequently, the product from the 1st round was diluted and used as a template for the 2nd round of PCR with primers R16F2n/R16R2 for 16Sr RNA and secAfor2/secArev3 for the secA gene (Gundersen and Lee 1996; Deng and Hiruki 1991; Hodgetts et al. 2008). This nested-PCR approach yielded distinct bands, approximately 1.2 kb (16Sr RNA) and 480 bp (secA), from the DNA of all ten symptomatic plants and positive sample, while no bands were observed in any of the asymptomatic plants. The nested PCR products were sequenced by BBS labs (Barcode biosciences, Bengaluru). The 16Sr RNA gene sequences, showing 100% similarity, were submitted to NCBI as representative sequences (Acc. No. OQ690013, OQ690014, OQ709133, OQ709134). Similarly, secA gene sequences were submitted with Acc. Nos. OR604283-86. BLAST analysis revealed a maximum 99.76% identity with Onion yellows phytoplasma and 'Ca. P. asteris' reference strain for 16Sr RNA gene, and a maximum 100% identity with 'Elaeis guineensis' stunt phytoplasma for secA gene. The phylogenetic tree constructed using the 16S rRNA and secA gene sequences indicated that the pea phytoplasma strains of this study clustered with 'Ca. P. asteris' (16SrI-B) related strains (Fig 2a and 2b). Additionally, the 16S rRNA sequences from this study, when subjected to Virtual RFLP using iPhyclassifier (Zhao et al. 2009), exhibited a pattern (Fig. 3) matching the reference pattern of the 16S group I, subgroup B (GenBank accession: M30790), with a similarity coefficient of 1.0. Previously, Rao et al. (2021) reported several crops associated with the 16SrI ribosomal group, including eight sub-groups from India. However, this report represents the first instance of a phytoplasma 16SrI-B group associated with phyllody and witches'-broom symptoms in pea, both in India and globally. Considering the economic importance of pea as a vegetable crop, the observed disease incidence and affected area are significant. Urgent attention is required to conduct additional research and implement preventive measures to avert the potential outbreak of this disease in the near future.

First Report of Candidatus Phytoplasma asteris (16SrI-B Subgroup) associated with phyllody disease of moth bean in world.

Moth bean (Vigna aconitifolia), a drought and heat-resistant legume from the Fabaceae family, is commonly cultivated in arid and semi-arid regions of the Indian subcontinent In September 2022, phyllody symptoms (Figure 1) were observed on 50-days-old moth bean plants at the ICAR-NBPGR research farm in Jodhpur, Rajasthan, India. The disease incidence ranged from 10 to 25%. To investigate the cause, ten symptomatic VacoJod (1-10) and ten asymptomatic VacoJod (11-20) Vigna aconitifolia plants were collected. Insect populations were also collected from the vicinity using the sweep-net method to examine the role of insect vectors. The leafhopper was identified based on morphological characterization as Empoasca sp. at the Division of Entomology, ICAR-IARI, New Delhi. DNA was extracted from midribs of all collected plants and the Empoasca sp., using Qiagen DNeasy Plant Mini Kit and Blood and Tissue kit, respectively. Nested polymerase chain reaction (Nested-PCR) with universal primers P1/P7 and R16F2n/R16R2 (Deng and Hiruki, 1991; Gundersen and Lee, 1996), and secA gene primers (secAfor1/secArev3 and secAfor2/secArev3) (Hodgetts et al., 2008) were employed to determine phytoplasma species association. Out of the 10 symptomatic plants and 10 leafhopper samples, 6 leafhopper samples and all symptomatic plants produced expected band sizes for the 16S rRNA (approximately 1.25 kb) and secA gene (480 bp). The PCR products were cloned, sequenced, and sequences (two each from moth bean and leafhopper) were submitted to NCBI GenBank with accession numbers OP941130, OP941132, OP941133 and OP941134 for 16S rRNA and OP958868, OP958869, OP958870, and OP958871 for secA gene sequences. Nucleotide BLAST analysis of 16S rRNA sequences revealed a minimum of 99.92% similarity with 'Primula acaulis' yellows phytoplasma (KJ494340) from Czech Republic. All 100% hits corresponded to 16SrI-B group phytoplasmas, for example rapeseed phyllody phytoplasma (CP055264) from Taiwan. Similarly, nucleotide BLAST analysis of secA sequences revealed a minimum of 99.15% sequence similarity with Paulownia witches'-broom phytoplasma (secA) (OP124308) from China. All 100% hits were of 16SrI-B group phytoplasmas, for example Ageratum conyzoides yellowing phytoplasma (MW401697, secA) from India. Phylogenetic analysis using MEGA11 (Tamura et al., 2021) clustered the moth bean and Empoasca sp. phytoplasma strains with 16SrI-B phytoplasma reference strains. iPhyClassifier tool classified the 16S rRNA gene sequences into 16Sr group I, subgroup B, with a similarity coefficient of 1.0 (Figure 2a, 2b). This marks the first report of the association of 'Ca. P. asteris' 16SrI-B related phytoplasma strain with moth bean plants globally. The 16SrI-B phytoplasma strain is prevalent in various crops in India (Singh et al., 2023). This report emphasizes the epidemiological studies and highlights the need for further research and preventive measures to manage the spread of this phytoplasma strain, which could impact crop production and food security in hot and dry regions.

First report of a 'Candidatus Phytoplasma aurantifolia'-related strain associated with faba bean phyllody symptoms in India.

Vicia faba L. commonly known as broad bean or faba bean is one of the most widely grown protein rich legume crops. Out of more than 50 faba bean-producing countries, about 90% production is concentrated in the Asian, European Union (EU), and African region (FAO, 2020). Owing to its high nutritional value, both the fresh pods and dry seeds are consumed. During March 2022, some plants with little leaf and phyllody symptoms such as leaf-like floral structures were observed in the experimental fields of Indian Agricultural Research Institute (IARI), New Delhi (Fig. 1 a, b, c). The twig samples were collected from two individual symptomatic and one from asymptomatic plant. DNA was extracted using CTAB (cetyl trimethyl ammonium bromide) method (Ahrens and Seemüller, 1992; Marzachi et al. 1998) and examined for the association of phytoplasma through nested PCR using the universal primers P1/P7 and R16F2n/R16R2 targeting the 16SrRNA gene (Deng and Hiruki 1991; Gundersen and Lee 1996) and the other set of primers secAfor1/secArev3 and secAfor2/secArev3 targeting secA gene (Hodgetts et al. 2008). The DNA from symptomatic plants resulted the amplicons of 1200bp and 840bp specific to 16S rRNA and secA gene respectively. The gel purified PCR products were cloned into pGEM®-T Easy Vector system (Promega) and outsourced for Sanger sequencing at Agri Genome Labs, Kerala, India. The resultant 16S rRNA sequences (GenBank Acc. No. OP978231, OP978232) and secA sequences (ON715392 and ON715393) were examined through NCBI BLASTn analysis. The 16S rRNA sequences of the V. faba strains shared a minimum of 99.85% similarity with the phytoplasma strain causing little leaf and phyllody disease of sesame in India (MW622017) and a maximum of 100% sequence identity with the Vigna radiata phyllody and necrosis phytoplasma strain of Jodhpur (OP935760) India, whereas the secA gene sequences showed 100% identity with Tephrosia purpurea witches'-broom phytoplasma (MW603929) from China and a minimum of 91.14% similarity with 'Candidatus Phytoplasma aurantifolia' (MW020541) from India. The pairwise comparison results were completely in support of the corresponding phylogenetic sequence analysis results of 16SrRNA and secA gene sequences of faba bean strains in comparison with other strains retrieved from GenBank database, wherein the faba bean strains got clustered with 16SrII-D subgroup related strains (Fig. 2 a and b). Virtual RFLP analysis through iPhyClassifer tool through in silico digestion of R16F2n/R2 region of 16S rRNA gene of the faba bean strain using 17 restriction endonuclease enzymes resulted in the RFLP profiles similar to that of the profile of phytoplasma subgroup 16SrII-D (Y10097: papaya yellow crinkle) used as reference strain with a similarity coefficient value of 1.0. All the results of this investigation confirmed the association of 'Candidatus phytoplasma aurantifolia' (16SrII-D) with the diseased faba bean plants in this study. Previous reports of phytoplasma infecting faba bean include a group 16SrIII strain detected in Spain in 2004 (Castro and Romero), a subgroup 16SrII-D strain detected in Sudan in 2012 (Alfaro-Fernandez et al.), a group 16SrII strain detected in Saudi Arabia in 2014 (Al-Saleh and Amer), and subgroup 16SrIII-J strains detected in Egypt in 2014 (Hamed et al.) and in Peru in 2021 (Torres-Suarez et al.). To the best of our knowledge, these findings, document the first report of the association of 'Candidatus Phytoplasma aurantifolia' (subgroup 16SrII-D) with faba bean plants in India. This report necessitates further research on the status of distribution of this phytoplasma strain in other locations and hosts in the country so as to develop possible strategies to contain its further spread and management of the disease.

First Report of Candidatus Phytoplasma asteris (16SrI-B subgroup) associated with phyllody disease of fenugreek in the world.

Fenugreek (Trigonella foenum-graecum) is a leafy vegetable and spice crop, native to Indian subcontinent and Eastern Mediterranean region. Phytoplasma infection symptoms were observed in fenugreek at ICAR-National Bureau of Plant Genetic Resources Regional Station, Jodhpur and Agricultural Research Station Mandore Jodhpur, Rajasthan, India. The first appearance of phytoplasma suspected symptoms of little leaf was recorded after 50 days of sowing in the months of January 2022. The major symptoms recorded were virescence, phyllody, shoot proliferation, witches-broom, little leaf, yellowing and overall stunted growth in 146 germplasm accessions at NBPGR research farm, Jodhpur and one major commercially cultivated variety RMT 305 at Mandore Jodhpur. Ten samples from symptomatic and five samples from asymptomatic fenugreek plants were collected and processed for total DNA extraction using the Qiagen DNeasy plant mini kit (Germany). The extracted DNA was amplified using nested PCR assays with universal phytoplasma detection primers for 16S rRNA gene (P1/P7 and R16F2n/R16R2) and secA gene specific primers (SecAfor1/SecArev3 and SecAfor2/SecArev3) (Schneider et al. 1995; Gundersen and Lee 1996; Hodgetts et al. 2008). The amplicons of ∼1.25 kb with 16S rRNA and ∼480 bp with secA gene specific primers were amplified in all symptomatic fenugreek samples. In negative control (asymptomatic plants) no amplification was observed with either of gene specific primers in gel electrophoresis. PCR amplified products from the six selected positive samples (FPP-NBPGR-J-01 to FPP-NBPGR-J-04 and FPP-MND-01 to FPP-MND-02) of 16S rRNA and secA gene, were sequenced from both ends. Sequences were deposited in the NCBI GenBank with accession numbers ON756108-ON756113 for 16S rRNA gene sequences and ON745809 to ON745814 for secA gene sequences. BLAST analysis of 16S rRNA and secA sequences revealed 100% sequence identity among themselves and 99.95 to 100% sequence identity with the earlier reported phytoplasma strains of aster yellows group related phytoplasma strains (GenBank Acc. No. MN239504, MN080270) belonging to Ca. P. asteris (16SrI group). Further analyses of the 16S rRNA and secA gene-based phylogenetic tree and the iPhyClassifier-based virtual RFLP analysis of 16S rRNA gene study demonstrated that the phytoplasma associated with fenugreek phyllody belonged to 16Sr group I ('Ca. P. asteris') and subgroup B (GenBank accession AP006628), with similarity coefficient of 1.0. Earlier association of 16Sr-II-D subgroup (Ca. P. australasiae) with fenugreek as host was reported from Pakistan (Malik et al., 2020). To the best of our knowledge, this is the first report of a 'Ca. P. asteris', 16SrI-B subgroup related phytoplasma strain associated with fenugreek phyllody in the world. The 16SrI-B phytoplasma strain is a widely distributed strain associated with several agricultural and horticultural crops of India (Rao 2021). This is not only the first instance of fenugreek phyllody disease found in India, but also the first instance of fenugreek phyllody caused by 16SrI-B subgroup phytoplasma worldwide. This report has epidemiological significance and needs immediate attention, as fenugreek is one of the most common seed spice crop being grown all over India.

First report of 'Candidatus phytoplasma asteris' (16SrI) from Cassia fistula showing symptoms of flat stem and witches'-broom in India.

Cassia fistula commonly known as 'golden shower tree' is a deciduous tree with a greenish-gray bark and complex leaves with lovely clusters of yellow blossoms that is also utilized for several purposes in traditional medicine offer therapeutic characteristics (Pawar et al., 2017). Random spotting of flat stem symptoms along with unopened flower beds was observed in C. fistula plant during March 2022 in IISER (Indian Institute of Science Education and Research), Thiruvananthapuram, Kerala, India and during May 2022 in SKUAST (Sher-e-Kashmir University of Agricultural Sciences and Technology), Jammu, which were suggestive of phytoplasma infection (Fig. 1 a-e). Surge of leaf hoppers was also observed in and around the tree. The leaf samples were collected from 3 individual C. fistula trees showing suspected symptoms of phytoplasma and one sample from asymptomatic plant of both the states. Leafhopper (LH) species were collected using sweep net method from both the locations. DNA was extracted using CTAB (Cetyl trimethyl ammonium bromide) method and nested universal PCR primers P1/P7 and R16F2n/R16R2 for the 16S rRNA gene (Deng and Hiruki 1991; Gundersen and Lee 1996) and secAfor1/secArev3 and SecAfor2/ SecArev3 for SecA gene (Hodgetts et al. 2008) were employed for the analysis of the phytoplasma strain association. The symptomatic plants and leaf hopper species showed positive bands of 1.2kb and 480bp for 16S rRNA and SecA gene respectively along with. Purified PCR products of both the genes (16Sr RNA and sec A) were ligated into pGEM ®T vector and cloned in Escherichia coli (DH5-α) were sequenced at Agri Genome labs, Kerala, India. The comparative sequence analysis using the BLASTn tool results showed 16S rRNA sequences acquired from plant samples (GenBank Acc. No. OP950857, OP950858) and the leafhoppers Hishimonus phycitis (OP538583) and Orosius albicinctus (OP538584) of Kerala had the minimum of 99.84% of similarity with Bitter gourd little leaf phytoplasma from Myanmar and maximum sequence identity (100%) with the Rapeseed phyllody phytoplasma strain from Taiwan. The sequences of phytoplasma strains from Jammu trees (Genbank Acc. No. OP801671 & OP801672) and H. phycitis (OP801673) shared 100% similarity with each other as well as with North American grapevine yellows and a minimum of 97.65% with Beta vulgaris phytoplasma from Poland. The pairwise comparison results were completely supported by the corresponding phylogenetic sequence analysis of 16S rRNA and SecA gene sequences of all the isolates in the study which clustered with 16SrI-B subgroup related strains. Virtual RFLP analysis through iPhyClassifer results that were derived from in silico digestions of R16F2n/R2 region of 16S rRNA gene using 17 restriction endonucleases enzymes indicated that all the samples produced similar virtual RFLP profiles identical to the reference strain of 16SrI-B phytoplasma subgroup (aster yellows: Acc. No. M30790) with a similarity coefficient value of 1.0. To the best of our knowledge, this is the first report of the phytoplasma association of 'Ca. P. asteris' (16SrI-B) subgroup with Cassia fistula in the world.

First report of association of 'Candidatus Phytoplasma asteris' with Moringa oleifera leaf yellowing and stunting disease in India.

Moringa oleifera (family Moringaceae) also known as the 'drumstick tree' is a significant nutritious and medicinal plant that is commonly grown in India and contains a variety of vital phytochemicals. M. oleifera is used in several Indian herbal medicine formulations to treat a variety of illnesses (Kumar and Rao 2021). Typical phytoplasma symptoms of leaf yellowing and stunting were observed in M. oleifera trees up to 10% incidence at Acharya Narendra Dev University of Agriculture & Technology, Ayodhya, Uttar Pradesh, India in November 2021 and stunting with less fruit bearings symptoms with 8% incidence in October 2021 at Jonnalakothapalle village of Mudigubba mandal of Ananthapuramu district in Andhra Pradesh, India (Fig.1a, b). To investigate the possibility of a phytoplasma association with the symptoms, total DNA was isolated from the leaf samples collected from two diseased and two healthy plants from both the locations using CTAB method. The DNAs isolated were analysed by nested polymerase chain reaction (PCR) with universal phytoplasma primer pairs P1/P7 and R16F2n/R16R2 for the 16S rRNA gene (Deng and Hiruki 1991; Gundersen and Lee 1996) and secAfor1/sArev3 and SecAfor2/ SecArev3 for secA gene (Hodgetts et al. 2008). Amplicons of the expected size (~1.25kb from 16S rRNA gene and ~480bp from secA gene) were obtained from symptomatic plants only. The nested PCR products were cloned (pGEM-T Easy Vector, Promega), sequenced (ABA Biotech, India) and the sequences were deposited in GenBank with accession numbers OP358449, OP358450, OP358451, OP358452 for the 16SrRNA gene (~1.25 kb) and OP358443, OP358444, OP358445, OP358446 for the secA gene (~480 bp). BLASTn analysis revealed that the partial 16S rRNA gene sequences of M. oleifera phytoplasma isolate shared up to 99.9% sequence identity with the strain 'Candidatus Phytoplasma asteris' (Accession numbers MN909051, MN909047) and secA gene sequences shared up to 100% sequence identity with 'Ca. Phytoplasma asteris' (Accession numbers KJ434315, KJ462009) belonging to 16SrI group. The 16S rRNA and secA genes sequence-based phylogenetic analysis (Figure 1d,e) showed that the phytoplasma strain associated with M. oleifera leaf yellowing and stunting clustered within the 16SrI phytoplasma group closest to 16SrI-B ('Ca. P. asteris') subgroup strains. Furthermore, the virtual RFLP pattern derived from the query 16S rDNA F2nR2 fragment is identical (similarity coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank accession: AP006628). To the best of our knowledge, this is the first report of the 16SrI-B subgroup of the phytoplasma strains with M. oleifera in the world. 'Candidatus Phytoplasma asteris' (16SrI-B subgroup) strains have been reported from several other commercial crops and weed hosts in India and efficient leafhopper vectors have been identified (Rao 2021; Reddy 2021). This indicates that the 'Ca. P. asteris'-related strains (16SrI-B) are widespread and infecting several plant species in India. The increasing incidence of the 16SrI-B strain and its wide host range in India strongly suggests further research into the epidemiology involved in the dynamic spread of the disease in order to recommend a suitable management approach.

Transcriptomic analysis of bakanae disease resistant and susceptible rice genotypes in response to infection by Fusarium fujikuroi.

BACKGROUND: Fusarium fujikuroi causing bakanae is one of the most significant pathogens of rice and much responsible for yield losses thereby emerging as a major risk to food security. METHODS: In the present study transcriptomic analysis was conducted between two contrasting resistant (C101A51) and susceptible (Rasi) genotypes of rice with the combinations of C101A51 control (CC) vs. C101A51 inoculated (CI); Rasi control (RC) vs. Rasi inoculated (RI) and C101A51 inoculated (CI) vs. Rasi inoculated (RI). RESULTS: In CC vs. CI commonly expressed genes were 12,764. Out of them 567 (4%) were significantly upregulated and 1399 (9%) genes were downregulated. For the RC vs. RI 14, 333 (79%) genes were commonly expressed. For CI vs. RI 13,662 (72%) genes were commonly expressed. Genes related to cysteine proteinase inhibitor 10, disease resistance protein TAO1-like, oleosin 16 kDa-like, pathogenesis-related protein (PR1), (PR4), BTB/POZ and MATH domain-containing protein 5-like, alpha-amylase isozyme were upregulated in resistant genotype C101A51. Whereas, genes related to GDSL esterase/lipase, serine glyoxylate aminotransferase, CASP-like protein 2C1, WAT1-related protein, Cytoplasmic linker associated proteins, xyloglucan endotransglucosylase/hydrolase protein and ß-D xylosidase 7 were upregulated in susceptible genotype Rasi. Gene ontology analysis showed functions related to defence response (GO:0006952), regulation of plant hypersensitive type response (GO:0010363), Potassium ion transmembrane activity (GO:0015079), chloroplast (GO:0009507), response to wounding (GO:0009611), xylan biosynthetic process (GO:0045492) were upregulated in resistant genotype C101A51 under inoculated conditions. CONCLUSION: Real time PCR based validation of the selected DEGs showed that the qRT-PCR was consistent with the RNA-Seq results. This is the first transcriptomic study against bakanae disease of rice in Indian genotypes. Further, functional studies on identified genes and their utilization through different methodology will be helpful for the development of bakanae disease management strategies.

Management of bakanae disease of rice using biocontrol agents and insights into their biocontrol mechanisms.

Bakanae disease is an emerging problem for the Basmati rice cultivation in India. Forty-seven endophytes isolated earlier along with three Talaromyces flavus isolates evaluated against Fusaium fujikuroi [Nirenberg] bakanae pathogen [isolate F250] through dual culture and enzymatic assays. Out of 50 isolates, 6 isolates namely, Tf1, Tf2, Tf3, Fusarium equiseti, Fusarium sp. and Trichoderma sp. produced good inhibitory results under in vitro conditions and were proceeded with in planta studies and conducted microscopic studies and real-time PCR assays. Microscopic studies revealed that the defense response system of plants was activated to a longer extent in bioagent treatments, since the number of live nuclei (DAPI staining) and green stained live plant cells (FDA staining) were higher as seen in treated plants when compared to pathogen-inoculated and uninoculated control when observed under confocal laser scanning microscopy. The analysis of cell cycle-related genes expressed during the ROS activity showed increased expression of the cell cycle-related genes involved. The selected isolates were also tested under glasshouse for disease inhibition studies. F. equiseti, Fusarium sp. and Trichoderma sp. gave a disease inhibition of, 87%, 66% and 94%, respectively. Tf2 and Tf1 isolate dominantly inhibited the disease with 95% whereas Tf3 also inhibited successfully with 70%. Through the results of our study, we can deduce that the T. flavus (Tf1, Tf2, Tf3) isolates and the endophytes F. equiseti, Fusarium sp. and Trichoderma sp. may represent an important biocontrol agent to control the bakanae disease of rice and also implicated that could further be befitting to capitalize them for field evaluations.

Draft genome sequence of 'Candidatus Phytoplasma australasia', strain SS02 associated with sesame phyllody disease.

'Candidatus Phytoplasma' is an uncultivated, intracellular bacterial plant pathogen transmitted by phloem-feeding insect vectors. Among the group of phytoplasmas, the Peanut Witches' Broom or 16SrII group of phytoplasmas associated with various diseases cause severe crop losses every year in India. The 'Ca. Phytoplasma sp.' strain SS02 was associated with phyllody disease of sesame plants collected from New Delhi. The genome sequence of strain SS02 was obtained using its genomic DNA enrichment and hybrid assembly of sequences generated on Illumina and Oxford Nanopore Technologies MinION platforms. The hybrid assembly strategy generated a draft genome with 60 contigs totaling 553,228 bp of length with more than 400 × depth coverage and 95.21% of the estimated completeness. The SS02 genome draft sequence contains 465 protein-coding genes, 17 tRNA genes, and 3 rRNA genes. The availability of this draft genome also provided a foundation for genome-scale genotypic analyses.

Whole Genome Sequencing of Fusarium fujikuroi Provides Insight into the Role of Secretory Proteins and Cell Wall Degrading Enzymes in Causing Bakanae Disease of Rice.

Fusarium fujikuroi causing bakanae disease has emerged as one of the major pathogen of rice across the world. The study aims to comparative genomic analysis of Fusarium fujikuroi isolates and identification of the secretary proteins of the fungus involved in rice pathogenesis. In the present study, F. fujikuroi isolate "F250" was sequenced with an assembly size of 42.47 Mb providing coverage of 96.89% on reference IMI58289 genome. A total of 13,603 protein-coding genes were predicted from genome assembly. The average gene density in the F. fujikuroi genome was 315.10 genes per Mb with an average gene length of 1.67 kb. Additionally, 134,374 single nucleotide polymorphisms (SNPs) are identified against IMI58289 isolate, with an average SNP density of 3.11 per kb of genome. Repetitive elements represent approximately 270,550 bp, which is 0.63% of the total genome. In total, 3,109 simple sequence repeats (SSRs), including 302 compound SSRs are identified in the 8,656 scaffolds. Comparative analysis of the isolates of F. fujikuroi revealed that they shared a total of 12,240 common clusters with F250 showing higher similarity with IMI58289. A total of 1,194 secretory proteins were identified in its genome among which there were 356 genes encoding carbohydrate active enzymes (CAZymes) capable for degradation of complex polysaccharides. Out of them glycoside hydrolase (GH) families were most prevalent (41%) followed by carbohydrate esterase (CE). Out of them CE8 (4 genes), PL1 (10 genes), PL3 (5 genes), and GH28 (8 genes) were prominent plant cell wall degrading enzymes families in F250 secretome. Besides this, 585 genes essential for the pathogen-host interactions were also identified. Selected genes were validated through quantitative real-time PCR analyses in resistant and susceptible genotypes of rice at different days of inoculation. The data offers a better understanding of F. fujikuroi genome and will help us enhance our knowledge on Fusarium fujikuroi-rice interactions.