Molecular Characterization of Fusarium and Screening of Cowpea [Vigna unguiculata (L.) Walp.] Germplasms against Fusarium Wilt (Fusarium oxysporum f. sp. Tracheiphilum)

Being short gestation, low input and resilience crop, cowpea in farming system becomes an innovative approach for climate change, soil degradation and nutrient depletion. Having originated in Africa, where the large genetic diversity of wild types occurs spreads into all continents, now mainly grown in many parts of Asia, Europe, USA, Central America and South America (Tetteh et al., 2020). Adaptability of the cowpea in all over is due to its high resilience to harsh conditions, especially hot and dry environments and poor soils (Boukar et al., 2018). Cowpea plays multifunctional traits, including food for human consumption as grains, tender leaves and pods; maintains soil-ecology balance through nitrogen fixation by facilitating symbiosis with nodulating bacteria; and provides fodder for livestock, it also helpful as a cover crop or an erosion-preventing crop; it aids in suppressing weeds; supports in the retention of moisture and contributes significant economic productivity and environmental sustainability (Omomowo and Babalola, 2021).
       
Fusarium wilt caused by fungal pathogen, Fusarium oxysporum f. sp. tracheiphilum (fot), is one of the soil-borne diseases that cause major threat in many of the cowpea growing area; substantially reduces the yield of 30 to 100% with high plant mortality and severe problem in cowpea production (Pottorff et al., 2014). The occurrence and epidemic spread of the pathogen depends on the soil factors like nutrient status, temperature (25-28°C), moisture and resistant varieties. The pathogen invades into the plant through roots and enters the vascular system; infected plants exhibit leaf chlorosis, necrosis, wilting, vascular discoloration and death of the plant with severe economic loss (Omoigui et al., 2018). Symptoms of fusarium wilt begins with sudden yellowing of foliage to defoliation; at the severe conditions, the lower portion and upper part of the tap root system together forms as swollen like structure and slowly became shredded and disintegrated (Senthilkumar, 2003). Wide range of host plants including family members of Leguminosae, Malvaceae and Solanaceae and the presence of dormant thick-walled asexual chlamydospore, which germinates by the induction of plant root exudates, in the soil crust are the major constraints to control the fusarium pathogen completely. Though it is seed and soil borne pathogen, is very difficult to manage using fungicide alone; and continues usage of fungicide for soil fumigation, mainly methyl bromide leads to environmental damage as well as occurrence of resistant strains of pathogen (Omoigui et al., 2018). So, the use of resistant varieties is one of the cost effective and environmentally safe method to control fusarium wilt in cowpea. Hence, the study aimed at identification and characterization of causal organism and screening of cowpea genotypes to identify resistant against fusarium wilt.

Present study was conducted at Department of Plant Breeding and Genetics, College of Agriculture, Vellayani during 2019-20, for molecular characterisation of pathogen causing fusarium wilt in cowpea and screening of cowpea varieties against the same for disease resistance breeding programme.
 
Isolation, identification and multiplication of Fusarium oxysporum spp.
 
The infected cowpea plants (Vigna unguiculata L.) were collected from field, stem and roots were washed in running water to remove adhered soil particles. The infected collar portion and root bits were cut into small pieces of 1-2 mm size and surface sterilized with 0.1% mercuric chloride (HgCl₂) followed by three times washes with sterile water. The 4-5 small bits were transferred aseptically into a petridish containing solidified Potato Dextrose Agar (PDA) amended with streptomycin sulphate and tetracycline hydrochloride to minimize chances of bacterial growth. Plates were incubated at 28°C and undergone periodic observation. From 3 days old culture plates, 5 mm disc of fungus was sub-cultured into fresh 15ml of sterilized and solidified PDA, incubated for 3 days at 28±2°C. The mycelial growth, colony characters and spore specifications were recorded three days after inoculation (DAI) on daily basis. The pathogen was characterized according to the epitypification given by Lombard et al., (2019) under microscope. Isolated fungus was multiplied in sand maize medium, which was prepared by mixing 90 g of riverbed sand and 10 g of maize meal (9:1) with 20 ml of distilled water in 250 ml of Erlenmeyer flask; and sterilized the mix at 15 lb for 20 minutes. After cooling, total of 10 bits of fungal culture along with PDA was aseptically transferred into the flask and closed air tightly; incubated for 15 days at room temperature (28±2°C) for multiplication.
 
Genomic DNA extraction, PCR amplification and nucleotide  sequencing
 
The genomic DNA of pure fungal isolates was extracted using NucleoSpin® Plant II Kit. The quality of the DNA isolated was checked using agarose gel (0.8%) electrophoresis. The gels were visualized in a UV transilluminator (Genei) and the image was captured under UV light using Gel documentation system (Bio-Rad). The ITS ribosomal DNA regions were amplified by PCR using universal primer pairs, ITS1-ITS4. PCR reaction mixture contained 200 ng fungal genomic DNA, 5 µl of 2×Phire Master Mix, 4 µl distilled water and 0.25 µl of each primer. Amplification was done in a Gene Amp-9700 thermal cycler (ABI, USA). Sequencing reaction was done in a PCR thermal cycler (GeneAmp PCR System 9700, Applied Biosystems) using the BigDye Terminator v3.1 Cycle sequencing Kit (Applied Biosystems, USA).
 
Sequence analysis
 
The sequences were trimmed, assembled and consensus sequences were obtained using BioEdit software. The obtained consensus sequences were deposited in NCBI database under accession number MZ706473. The obtained nucleotide sequence was searched through BLASTN at GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/) to compare with ITS reference sequence available in the database. Multiple sequence alignment of ITS sequence with reference sequence from gene bank was performed using Clustal W available in BioEdit Software. Phylogenetic evolutionary relationship analysis was performed using Molecular Evolutionary Genetic Analysis software (MEGA 7) by Neighbor-joining method with 1000 bootstraps run.
 
Screening of cowpea genotypes against Fusarium oxysporum spp.
 
Seedling screening was conducted in completely randomised design with three replications along with control during 2019-20. A set of 115 cowpea genotypes consisting of germplasm from NBPGR, New Delhi, AVRDC, Taiwan, Dept. of Plant Breeding and Genetics, College of Agriculture, Vellayani and released varieties in Kerala Agricultural University (KAU) were collected for the study. The Fusarium isolate multiplied in sand maize medium (9:1) were incorporated to the sterilized soil at the rate of 10% (w/w). One week old cowpea seedling from each genotype was transplanted to the soil containing Fusarium and a control plant were also maintained and monitored regularly. The number of days to wilt, disease intensity, disease scoring and disease reaction were assessed visually and weekly, starting at 20 days post inoculation. The disease intensity (Chattopadhyay and Sen, 1996) was calculated by using the formula:

The culture had medium growth rate (5 mm/day), when it was cultured aseptically in PDA media from infected stem (Fig 1) Maximum radial growth and growth parameters with abundant sporulation were noticed in Fusarium spp. grown on potato dextrose agar (Gupta et al., 2010). Fusarium oxysporum spp. is an anamorphic species circumscribed by some morphological criteria, basically shape of macroconidia, structure of microconidiophore and formation and disposition of chlamydospores. The colonies of isolate were appressed to floccose in texture, white on the upper surface, reddish brown or faint pink on the lower side of the petridish (Fig 2). Some species of fusarium produces no colour, while others usually produce pale purple to dark magenta pigment on agar media (Janevska and Tudzynski, 2018), but there is no relationship between pathogenicity and pigments produced (Lombard et al., 2019). The conidiophores consisted of monophialides on aerial mycelium and produces asexual spores such as microconidia, macroconidia and chlamydospores, which lacks sexual reproduction. Microconidia were aseptate, abundant, oval to ellipsoidal, cylindrical, straight or curved form and 12.879×3.570 µm in size. Aggregates of aseptate microconidia forms false head structure was a characteristic feature of Fusarium oxysporum spp. Microconidia produced from intercalary phialides in false heads (Ohara and Tsuge, 2004). The macroconidia were 3-5 septate, thin walled, fusoid, pointed ends, occasionally falcate with terminal cell, hooked, pedicellate basal cell and 32.409×5.297 µm in size. Chlamydospores when present were terminal and intercalary in position (Fig 3). The comparison of isolate with already characterized fusarium pathogen by Lombard et al., (2019) was depicted (Supplementary Table 1).







@supplymentarytable1
       
The ITS region contains multiple tandem repeats of ribosomal RNA in the haploid genome, which are very useful in species identification, hence it has been considered as a standard marker in DNA bar-coding of fungal species (Janevska and Tudzynski, 2018). Based on the assembled sequences from both forward and reverse primers, the obtained ITS sequences containing ITS1-ITS4 deposited in the gene bank at NCBI (accession number MZ706473) (Fig 4). Basic Local Alignment Search Tool (BLAST) analysis showed that the assembled sequence had high percentage of similarity index (99-100%) and query recovery (100%) with the Fusarium oxysporum reference sequences (Supplementary Table 2). Phylogenetic analysis revealed that the isolate is less distant and diverse from Fusarium oxysporum. The phylogenetic tree divided into clades and subclades, in which the isolate was related to potential Fusarium oxysporum (OL679453.1) strain supported by 52% bootstrap (Fig 5).



@supplymentarytable2



Cowpea accessions were abundantly available with huge diversity in characters, which could be useful in the improvement programme and in developing varieties with specific traits (Nalawade et al., 2021). Screening of cowpea germplasm against fusarium wilt resistance are not done so far. The screening of 115 cowpea germplasm collected from different sources were resulted in 18 resistant (R) genotypes with 0% incidence of symptoms, these genotypes need to be further evaluated for the fusarium linked characters under sick plot at field level. Remaining 26 were moderately resistant (MR) with 0-25% incidence of symptoms, 31 were moderately susceptible (MS) with 26-50% incidence of symptoms, 31 were susceptible (S) with 51-75% incidence of symptoms and 9 genotypes were highly susceptible (HS) with >75% incidence of symptoms (Table 1) (Fig 6). Artificial screening of many crops against fusarium wilt reaction were done and scored accordingly in chickpea (Kumar et al., 2019), lentil (Roy et al., 2021). Single dominant gene with lack of maternal effect and linkage; controls the resistant reaction in cowpea against fusarium wilt (Omoigui et al., 2018). The dominant inheritance Fusarium wilt resistance were also observed in pigeon pea (Singh et al., 2018). So, the crop improvement for resistant breeding through the marker assisted selection become uncomplicated and easily recognizable; which can shorten the breeding cycle.


       
The wilting symptom began after 20 days up to 53 days of inoculation in all genotypes, which means the disease severity was more during flowering or reproductive stage than seedling stage; and incidence of disease varied greatly within the genotypes. Late wilt percentage was greater than early wilt percentage in all the genotypes of chickpea under pot culture screening than sick plot method (Yadav and Kumar, 2019). Disease intensity were ranged from 0-80%, with reaction of resistant and highly susceptible respectively. The eighteen cowpea genotypes identified as resistant against fusarium wilt could be used as donor parents for wilt resistance in resistant breeding programme.

Fusarium wilt in cowpea causes tremendous decline in the cowpea production, and the management cannot be done completely due to is soil-born nature. It causes yellowing, necrosis, vascular discoloration, basal swelling, and fully distraction of the plant. The pathogen produces asexual spores like aseptate microconidia, 3-5 septate macroconidia and thick walled, dormant chlamydospores, without sexual reproduction. The wilting symptom in cowpea seedlings under artificial screening, began after 20 days up to 53 days of inoculation in all genotypes, with more disease severity during flowering or reproductive stage than seedling stage; and incidence of disease varied greatly within the genotypes. The resistant cowpea varieties identified in the study might be used as donor parents for resistant breeding programme.

The authors wish to thank financial contributions from the Department of Science and Technology (DST) as INSPIRE Scholarship and Kerala Agricultural University (KAU). We are thankful to AVRDC, Taiwan, NBPGR, New Delhi and KAU, Thrissur, for providing cowpea germplasms for the study. We also thank Integrated Farming Systems Research Station (IFSRS), Karamana for providing laboratory facilities.

The authors declare no conflicts of interest.