Morpho-molecular identification and first report of Fusarium equiseti in causing chilli wilt from Kashmir (Northern Himalayas)

Chilli (Capsicum annuum L.) is one of the most significant vegetable and spice crop. Wilt caused by Fusarium Sp. has emerged as a serious problem in chilli production. Internal transcribed spacer (ITS) region is widely used as a DNA barcoding marker to characterize the diversity and composition of Fusarium communities. ITS regions are heavily used in both molecular methods and ecological studies of fungi, because of its high degree of interspecific variability, conserved primer sites and multiple copy nature in the genome. In the present study we focused on morphological and molecular characterization of pathogen causing chilli wilt. Chilli plants were collected from four districts of Kashmir valley of Himalayan region. Pathogens were isolated from infected root and stem of the plants. Isolated pathogens were subjected to DNA extraction and PCR amplification. The amplified product was sequenced and three different wilt causing fungal isolates were obtained which are reported in the current investigation. In addition to Fusarium oxysporum and Fusarium solani, a new fungal species was found in association with the chilli wilt in Kashmir valley viz., Fusarium equiseti that has never been reported before from this region. The studies were confirmed by pathogenicity test and re-confirmation by DNA barcoding.

www.nature.com/scientificreports/ followed by the wilting of the whole plant which is caused due to the damage in vascular system thus hampering nutrient and water uptake [19][20][21] . Fusarium equiseti is considered to be a weak pathogen on cereals and is occasionally found to be associated with Fusarium head blight infected kernels 22 .This species is commonly found in tropical and sub-tropical areas 23 . The species is a pathogen to varied range of crops and it has been recently reported to be a causal organism of wilt in Capsicum chinense in Mexico 24 . The pathogenicity of this species is has underestimated though. The species belongs to the F.incarnatum-F. quiseti complex and it is genetically diverse. However, various investigations have been carried out on chilli wilt causing pathogens and several management strategies has been designed for controlling the chilli wilt. Fusarium species have several morphological characteristics that help these for distinct identification and one of the prominent features is development of various shapes and sizes of macro and micro conidia which are asexual spores. Other structures that they form are called chlamydospores spores 25 which ensure the survival of the pathogen in soil and in plant for many years thus making the management and control of the diseases caused by Fusarium species very difficult to cater 26,27 . These are also identified on the basis of growth rate on agar media and the pigmentations produced by them 28 . Moreover, the morphological identification can be quite difficult among the Fusarium species 29 . Identification of the species is done through macro and microscopic analysis but the most reliable form of identification is through the information gathered via nucleotide sequencing from conserved gene regions which include Internal Transcribed Spacer (ITS) 30 . The sequence information using ITS regions has been immensely used in phylogeny and taxonomy of Fusarium species 31 as ITS regions have been known to successfully aid in identification between the species 32 . ITS is differentiated into two regions ITS1 and ITS2 (genes 18S to 5.8S and 5.8S to 28S respectively) 33 . There are more than 172,000 fungal ITS sequences present in Genbank 34 .
A deep comprehension of the populations of pathogens is important as they show variations in pathogenicity, response to management systems, environment and host differences 35 . Thus population biology of the pathogen needs to be studied with depth. Since, the incidence of wilt is very prevalent in Kashmir so it is the need of an hour to do a detailed investigation of pathogen causing chilli wilt. It has not been yet cleared that among the population of Fusarium species how many are responsible for causing chilli wilt in Kashmir region. In this regard major chilli growing hotspots, pathogens association and prevalence of major pathogen in different areas of Kashmir Himalayas were investigated and morphological and molecular studies were carried out which, for the first time revealed Fusarium equiseti to be one of the causal organisms for chilli wilt in Kashmir along with Fusarium oxysporum, Fusarium Solani.

Materials and methods
Collection, Purification and Maintenance of culture. Survey of vegetable growing areas in four districts of Kashmir valley viz., Srinagar, Budgam, Pulwama and Anantnag was carried out to assess the prevalence of the disease ( Table 1).The fungi from the infected samples were isolated using tissue bit technique 36 . The isolated pathogens were purified using hyphal tip and / or single spore technique 37 . Pure cultures thus obtained were maintained by repeated sub-culturing at intervals of 45 days for further studies and the sterile cultures were stored at 4 °C in a refrigerator.
Identification and Pathogenicity test. Identification of the pathogen was confirmed on the basis of morphological and the pathological characteristics. Identification was confirmed from division of Plant Pathology, SKUAST-K and also confirmed through DNA barcoding. The pathogen inoculum was multiplied on sand meal agar medium. The medium was prepared by autoclaving 90 g dry sieved sand and 10 g maize meal with 40 ml of distilled water at 1.05 kg cm-2 pressure for half an hour for three consecutive days. The sterilized medium was then inoculated with respective fungi and incubated for three weeks at 25 ± 1 °C with daily shaking of flasks to get uniform growth. The inoculums thus prepared were added to the sterilized sand soil (2:1) potting mixture @ 10 per cent (w/w) by mixing it with upper layer of soil and allowed for 7 days to infest soil 38 (Raj and Singh 1973;Najar 2001). Forty five days old chilli seedlings (cv. 'Shalimar Long'), raised by growing surface sterilized seeds in sterilized sand soil (2:1) potting mixture were gently uprooted and transplanted in the sterilized

Morphological and cultural characteristics of the isolated pathogen(s). The morphological char-
acteristics of the causal pathogen(s) were studied in-vivo after culturing on artificial medium to identify the associated pathogen(s). The pathogen cultures were grown on potato dextrose agar (PDA) medium and the semi-permanent slides prepared from 10 days old colonies. The important characters studied were: • Hyphae, width, septation and colour • Conidia, shape, size and colour • Conidiophore, shape, size and colour • Chlamydospore, shape, size and colour.

DNA Extraction and PCR Amplification.
DNA extraction was carried out using 400 microlitre extraction buffer. This buffer is same as that described by Cenis et al. 39 . The genomic DNA of fungal isolates was loaded on 0.7% agarose in 1XTAE for 30 min. PCR amplification was carried out using ITS1 andITS2 primer pair for the amplification of all the three isolates. The primers were designed manually using, Oilgocalc, Clustalw software the primer pairs used for amplification were ITS1F Primer (5′CCT GCG GAG GAT CATTA 3′), ITS2R Primer (5′TCC TCC GCT TAT TGAT3′). PCR amplification for ITS1 5.8S-ITS2 region was carried out in a reaction volume of 25 µl in 0.2 ml PCR tubes. The amplification reaction was carried out in thermo cycler (Applied Biosystems) for 1-2 h for amplification of ITS1 -ITS2 region followed by gel electrophoresis procedure and compared with 100 bp DNA ladder, respectively ( Fig. 1).

Sequencing, Nucleotide Alignment and Phylogenetic Analysis.
Amplified PCR products were sequenced at Agri Genome Labs (Infopark Road, Kakkanad, Kerala, India). The same primers utilized for the PCR amplification were used for sequencing as well. PCR products (sequences) were assembled using the DNA Baser V.4 program to produce complete contigs. These were further aligned using the CLUSTAL W method (Bio-Edit). A search of homologous sequences was performed by BLAST analysis at NCBI (http://ncbi.nlm.nih. gov/BLAST ). The MEGA7 (Molecular Evolutionary Genomics Analysis Version 7) 40 constructed dendrograms from the 10 isolates from the current study and reference strain sequences from GenBank. There were 100 replications for every bootstrap value. For validation of results, an out group fungal pathogen Alternaria was selected.

Results
Diseased plant samples were collected from four districts of Kashmir region of India viz., Srinagar, Budgam, Pulwama and Anantnag on the basis of symptomatology, during 2018-2019. The characteristic symptoms discovered were lesions on the root/stem, brown tube-shaped structure discoloration, yellowing, weakening and death of the plants.

Molecular Characterization and phylogeny. After sequencing the PCR product and analyzing with
BLASTn, all sequences showed 98%-100% sequence homology with GenBank sequences. Sequences were submitted to NCBI GenBank and accession numbers were obtained (Table 1). Phylogenetic analysis revealed that our isolates clustered along with other submitted Fusarium isolates from GenBank. The sequences of Fusarium isolates formed same cluster with Fusarium equiseti, Fusarium solani and Fusarium oxysporum, but in separate subclusters. Alternaria formed a different cluster (outgroup) in the dendrogram (Fig. 9). We observed that using ITS region of the 10 isolates collected from different locations of J&K showed sequence homology with isolates reported from different regions particularly isolates of India, China, Egypt and Thailand.

Discussion
Chilli is a widely grown crop all over the world for its flavor and colour; and is believed to have many health beneficial properties like anti-inflammatory and antioxidant potential 41 . However, from past few years the production of chilli has been hampered due to many prevailing abiotic and biotic stresses which include many bacterial and fungal diseases 42 . Among the fungal diseases most dominating is Fusarium species to which 50-80% losses in the production are attributed 43  All the three isolates viz., F. oxysporum, F. solani, F. equiseti isolates evaluated within the present study were infective in nature with slight variation in virulence. The appearance of the specific symptom/disease severity varied depending on the pathogenicity or virulence level of specific isolate of fungus. The fungal pathogens isolated from wilted chilli plants, when artificially inoculated through rhizosphere inoculation technique on potted chilli plants exhibited typical disease symptoms. Koch's postulates were confirmed by re-isolating the pathogen from the artificially inoculated and infected plants. The plants developed initial symptoms on the second week of inoculation. The initial symptoms showed light green to yellowish discoloration of leaves followed by their shriveling, drooping and finally death of whole plant at fourth week of inoculation. The dried leaves remained clinged on the wilted plants. When the collar region of the plant was cut vertically, the vascular bundles showed brownish discoloration. The initial symptoms were first recorded in F.equiseti so, this strain of www.nature.com/scientificreports/ pathogen was more virulent as compared to other pathogens which were artificially inoculated (F.oxysporum, F.solani). F.oxysporum has also been reported from various regions of Kashmir valley and from many parts of India 49 F.solani has been also reported from many parts of India 50,51 . F. equiseti was shown to be prevelent in five different locations in Kashmir valley.
In the present studies, the internal transcribed spacer (ITS) amplification with genera and/or species specific ITS primers, clearly differentiated all the three isolates of fungi into F. oxysporum, F. solani, F. equiseti. In a recent study PCR when carried out on Fusarium found in Capsicum species using ITS regions, the sequences successfully resulted in identification of five Fusarium species as F. solani, F. oxysporum, F. equiseti, F. incarnatum, F. chlamydosporum showing dominance in F. solani and F. equiseti 52 . Based on amplification and size of the amplicons in different isolates, amplified with ITS primer combinations viz., ITS1F and ITS4Rdirected the amplification of ~ 500 bp ITS-rRNA uniform amplicons in all the isolates of F. oxysporum , F. solani, F. equiseti. These ITS primer mixtures, which targeted and amplified a specific gene/region within the internal transcribed Sequences were assembled, edited and multiple sequence alignments were performed using the ClustalW tool from MEGA 7.0 and a NJ tree, which was recommended as the standard barcoding method which was adopted and constructed using MEGA 7.0 software. In the present investigation three query sequences of different fungal species which were identified through sequencing were clustered together according to their similarity with their respective hits viz., Fusarium equiseti, Fusarium solani, Fusarium solani showing closeness with each other also.

Conclusion
In the present study, three isolates of fungi were collected and isolated from diseased root/stem samples of chilli the pathogenicity was demonstrated on very vulnerable chilli plants. The pathogenicity of the contagious spp. isolates was confirmed based on the capacity of each isolate to cause ailment and appearance of the particular side effects viz., yellowing, wilting or plant death. Morphological characteristics of the causal pathogen were studied both on host as well as on artificial culture medium to identify the associated pathogen. Gene sequencing studies of ITS1-5.8S-ITS2 gene were elucidated and all the sequences were submitted in GenBank (NCBI). The major chilli growing hotspots of Kashmir Valley were investigated in the current study. Among many pathogens F. oxysporum and F. solani and Fusarium equiseti were found to be prevalent in these areas. Fusarium equiseti incidence is reported for first time in Kashmir valley.   www.nature.com/scientificreports/