Cultural and Molecular Diversity of Fusarium pallidoroseum (Cooke.) Sacc Isolates Causing Wilt in Mothbean (Vigna aconitifolia)

The excessive reliance on major staple crops, climate change and degradation of cultivable land hinder agricultural sustainability, posing challenges to global food security and exacerbating rural poverty and malnutrition in developing nations. Thus, a shift towards alternative crops, specifically underutilized plant species, is crucial for enhancing crop diversity, resilience and nutritional security in the face of evolving pests and pathogens (Kanishka et al., 2023). Pulses are an integral part of many diets across the globe and they have great potential to improve human health (Kharte et al., 2022) and these crops are major source of protein in the vegetarian diet (Yadav et al., 2022). The moth bean (Vigna aconitifolia L.) is an underutilized legume crop belonging to the Fabaceae family, subfamily Papilionaceae. It is the one of the most drought tolerant Kharif crop species of pulse category (Kohakade et al., 2017). Moth bean is a multipurpose crop but mainly grown for food, feed, pasture and fodder (Sedani et al., 2021). The regular consumption of mothbean will reduce cardiac diseases, diabetes and obesity of human. Mothbean seed contain abundant antioxidant as well as polyphenol the level helpful for human health (Bhadkaria et al., 2022). It is known as orphan legume as well as superior nutritional quality legume which can be cultivated widely and has ability to grow across different eco-geographical regions of Asia particularly under the Indian subcontinent (Kanishka et al., 2023).

Vascular wilt caused by Fusarium pallidoroseum is common disease in moth bean. A pathogen’s greater adaptability strongly suggests the possibility of variations. Fusarium is most destructive soil borne fungus resulting into plant wilt and difficult to manage (Singh et al., 2019). The monocropping, cultivation of common variety/hybrids/genotypes leads to evolution in pathogen behaviour and infection. Pathogen develop physiological and genetical variation among itself than parent one (Baysal et al., 2013). Cultural, morphological and molecular variability in different pulses like chickpea, pigeon pea, lentil grown areas is in reports (Thaware et al., 2017; Reddy et al., 2022). Variability is always a desirable attribute of a disease because it facilitates in the pathogen’s survival in diverse niches and environments.The identification of variability helps in facilitating the development of effective strategies for managing multiple pathogenic variants (Arya et al., 2021). Hence, an investigation was conducted to study the cultural, morphological and molecular variability among the isolates of Fusarium spp. in moth bean growing areas of Bharuch district.

Isolation and pathogenicity of pathogen
 
The fixed plot survey was conducted during 2022 to study status of mothbean wilt under Bharuch district. The disease samples were collected from various locations of Vagra and Bharuch talukas of moth bean cultivating farmer’s field as per mentioned in Table 1. The collected samples were bought to the laboratory at Department of Plant Pathology, College of Agriculture, Navsari Agricultural University, Bharuch. The isolation of pathogen was done by tissue isolation method and was further purified by single spore isolation technique. The isolated pathogen of each location samples were consider as isolates during study. The Koch postulates of isolated culture causing moth bean wilt was proved by growing moth bean plants in pots containing pathogen-infested soil as per (Patel, 2022).


 
Cultural and morphological variability
 
It is essential to understand cultural and morphological variation among various isolates for better understanding of molecular variability of Fusarium causing moth bean wilt The Fusarium isolates causing mothbean wilt were used to study cultural variation on PDA medium. The cultural and morphological study conducted in Department of Plant Pathology, College of Agriculture, NAU, Bharuch, Gujarat in 2022. The cultural characters like mycelium colour, texture, margin, pigmentation and radial growth were observed. Euromex microscope (Made in Netherlands) was used for conidial measurements. Total ten observations of each character i.e. width of mycelium, size of conidia, conidial measurement and numbers of septa in macro and micro-conidia, chlamydospores size and position were recorded for were recorded and average of each character was calculated.
 
Molecular variability
 
Eleven isolates of Fusarium inciting mothbean wilt were molecularly characterized during March-2023 by using various six ISSR primers (Table 3) synthesized by Barcode Biosciences Pvt. Ltd. The experiment was conducted in the laboratory of Plant Pathology, College of Agriculture, Navsari Agricultural University, Bharuch. The procedure for fungal DNA isolation was followed as per (Murray and Thompson, 1980) which were modified in annealing temperature and primer concentrations. Amplification of DNA during PCR reactions using ISSR primers was accomplished by using reaction mixture.  [Template-2 µl (~50 ng of DNA), Primer-1 µl (~2.5 pmol), Master mix-7 µl, 10 picomole/µl oligonucleotide ISSR primers, 5U/μl Taq DNA polymerase, 10 X Taq buffer, 25 mM MgCI2, 10 mM DNTP. The PCR amplification of DNA for ISSR markers werecompleted in 35 cycles (Denaturation at 95°C for 30 sec, Annealing at 50°C for 30 sec and Extension at 72°C for 1 min). The analysis of the recorded data was done by counting of the reproducible and clear bands with binary code 1 and 0. Jaccard’s Similarity coefficient was generated using presence and absence of polymorphic bands with the use of NTSYS 2.20 Software. Based onsimilarity coefficient dendrogram was made by UPGMA (Unweighted Pair-Group Method with Arithmetical Averages). Jaccard’s similarity coefficient was calculated as per standard formula and Polymorphic information content (PIC) was calculated as per (Kumar et al., 2003).

Isolation and pathogenicity of pathogen
 
The isolation of pathogen was done by tissue isolation method and the Koch postulates of isolated culture causing moth bean wilt were proved in pot condition. The pot inoculated with pathogen recorded typical mothbean wilt symptoms i.e. drooping of leaves and cortical decay of seedling, the leaves turned yellow followed by wrinkling and drying, loss of turgidity with brownish discoloration of vascular system. The plant dies after seven days of pathogen inoculation. The results are similar with Patel (2022) found wilting of mothbean after seven days of pathogen inoculation.
 
Cultural and morphological characteristics
 
The variability of pathogen was studied by morphological and cultural basis for better understanding of molecular variability of Fusarium causing moth bean wilt. Total eleven isolates of Fusarium causing moth bean wilt were collected and isolated from Vagra and Bharuch talukas of Bharuch district (Table 1).

The cultural studies based on the mycelium colour, mycelial texture, margin, pigmentation and radial growth showed huge variation in the cultural characteristics of the Fusarium isolates. The mycelium colour varied from dull whitish, pinkish white, purplish white, bright white and dull yellowish white. The mycelial texture varied from thick profuse, thin profuse, thin compact, moderately thin compact and moderately thin profuse (Table 1). Similar results are in accordance with Yadav et al., (2022) who observed cottony colours among different isolates of F. verticillodies in maize. The radial growth was observed maximum (50 x 50 mm) in isolate SAFM-4 and minimum (35 x 30 mm) in isolate ARFM-1 (Table 1). This results were corroborates with finding of Venkataramanamma et al., (2021) who found texture of the mycelium was fluffy, appressed, partially appressed, fluffy and thread like in nature. Saxena et al., (2019) observed yellowish pigmentation and dark brown with creamy as well as dark tan culture of Fusarium oxysporum f. sp. lentis. The mycelial growth pattern was oppressed and fluffy. Purohit et al., (2017) observed that the colony diameter ranged from 2.8-9.7 cm.

The morphological studies based on size and breadth of conidia, number of septa in macro and micro-conidia and position of chlamydospores exhibited wide range of variation. The size of macro conidia of Fusarium isolates was ranged from 11.45 ´ 3.56 µm (BHFM-7) to 27.13 x 5.35 µm (BHFM-3) whereas in micro-conidia it was ranged from 7.23 x 3.02 µm (VAFM-6) to 15.59 ´ 5.14 µm (SA1FM-2) (Table 2). Septation in macro conidia ranged from 1-3 to 2-4. Isolate SA1FM-2 and RA2FM-5 recorded maximum (2-4) septation, followed by ARFM-1, BHFM-3, VAFM-6, BHFM-7, RA2FM-8, SA2FM-10 and ORFM-11 with (2-3) septation, tailed by SAFM-4 and ANFM-9 with (1-3) septation whereas in micro-conidia septation ranged from 0 to 1 (Table 2). The chlamydospores also showed remarkable variability in size and position of chlamydospores among the Fusarium isolates, the maximum (29.19 x 12.79 µm) was observed in isolate ORFM-11 and minimum (9.95 x 4.70 µm) in isolate BHFM-3 with the isolates showing either intercalary or terminal position (Table 2). Similar results are in accordance with Reddy et al., (2022) who found that the size of the micro-conidia ranged from 5.37 µm x 2.44 µm (Fsp-24) to 13.58 µm x 3.33 µm (Fsp-32) and the size of the macro conidia ranged from 16.62 µm x 3.35 µm (Fsp-35) to 39.05 µm x 4.61 µm (Fsp-33) and Hami et al., (2021) who observed septation in macro and micro-conidia, 3-4 and 0-1 respectively. 


 
Molecular variability
 
At the molecular level the 11 isolates of the pathogen with ISSR (6 primers) showed significant variation with 100 per cent polymorphism (Table 3). The amplified bands obtained were of various sizes, ranging from approximately 0.1 to 1.7 Kb. The amplicon size for (CAG) 5 ranged from 0.3 to 1.6 Kb. The minimum similarity coefficient obtained was 0.28 and maximum similarity coefficient obtained was 0.83 (Fig 1). The results of pairwise combination indicated the closest relationship between the isolates ARFM-1 and SAFM-4 i.e. 0.83 and the minimum similarity coefficient ormaximum diversity was observed in between isolates SAFM-4, BHFM-7 and ORFM-11 i.e. 0.28 with ISSR primers. The ISSR primers clustered 11 isolates at 48 per cent similarity level in the two major clusters (A and B) (Fig 1). Similar results are in accordance with Arya et al., (2021) who evaluated Twenty-two isolates at 62 per cent similarity level and were clustered into two major clusters (A and B), cluster A consisted nine isolates, further A and B clusters were divided into four sub-clusters.Clusters A consisted of eight isolates i.e. ARFM-1, (Aragama), SAFM-4 (Saran), RA1FM-8 (Rahad-I), BHFM-3 (Bhersam), RA2FM-5 (Rahad-II), SA2FM-10 (Sayakha-II), VAFM-6 (Vagra) and BHFM-7 (Bharuch) and cluster B included three isolates i.e. SA1FM-2 (Sayakha-I), ANFM-9 (Ankot) and ORFM-11 (Rahad-original). The results are similar with Navale et al., (2023) who evaluated the genetic diversity of Fusarium spp. in maize through twenty ISSR primers and five five major clades (I to V) and 10 to 12 sub-clades were generated during study and Nawade et al., (2017) studied molecular diversity of F. oxysporum f. sp. cumini by 11 ISSR primers and it categorized in to two main clusters. 

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The pure culture of pathogen causing mothbean wilt was sent for the identification to Indian Type Culture Collection (I.T.C.C.), Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi and it was identified as Fusarium pallidoroseum (Cooke.) Sacc (ITCC ID no. 11,584.21). Eleven isolates of Fusarium were isolated from moth bean growing areas of Bharuch district showed the maximum cultural, morphological and genetic variability within isolates. The maximum (50 x 50 mm) radial growth was observed in isolate SAFM-4 and minimum (35 x 30 mm) in isolate ARFM-1. The size of macro conidia of Fusarium isolates was ranged from 11.45 x 3.56 µm (BHFM-7) to 27.13 x 5.35 µm (BHFM-3) whereas in micro-conidia it was ranged from 7.23 x 3.02 µm (VAFM-6) to 15.59 x 5.14 µm (SA1FM-2). The molecular variability of 11 isolates of Fusarium causing mothbean wilt was studied by ISSR primers which showed significant variation with 100 per cent polymorphism.

All the authors declared that there is no conflict of interest.