Morpho-molecular and Pathogenic Variability of Wilt of Lentil from Indo-Gangetic Plains of India

Lentil (Lens culinaris (L.), 2n=2x=14) belonging to the family Fabaceae is an important legume food crop which is valued for its beneficial effects in improving soil fertility (Das et al., 2017). The seeds are rich in protein (30%), vitamins and minerals and also good source of nutrients like phosphorus, calcium and iron (Pal et al., 2016). The yield losses of lentil are mainly by insects and diseases range from 5 to 10% in temperate regions and 50 to 100% in tropical regions (Hiremani and Dubey, 2016; Mondal et al., 2021). Among the diseases affecting lentil, wilt caused by Fusarium oxysporum considered as one of the most devastating diseases which is responsible for its low productivity (Tosi and Cappelli, 2001, Sarwar et al., 2014). In India this disease may cause 5-10% yield losses but sometimes severe damage may result complete crop failure under favorable conditions for disease development (Chaudhary and Amarjit, 2002; Majumder et al., 2022). The major symptoms of the disease are curling of the leaves starting from the lower end and extending upwards, drooping and eventually death of the plant. The present study aimed at characterization of the F. oxysporum isolates collected from different lentil growing parts of West Bengal using morphological characters and aggressiveness.

Fungal cultures
 
Nine isolates of Fusarium oxysporum addressing nine lentil growing districts of West Bengal, India was collected, isolated by using PDA in BCKV, Pathology Laboratory, Division of Plant Pathology for the year of 2019-20. The fungal cultures were purified and single-spore cultures were maintained at 4°C on potato dextrose agar (PDA) medium and sub-cultured at periodical intervals.
 
Morphological variability
 
All 9 isolates of Fusarium oxysporum collected from different lentil growing districts of West Bengal, India (Das et al., 2022) was studied for their morphological variability. To study cultural characteristics, 5 mm mycelia bits of each isolate were taken from the actively growing cultures and centrally placed on 90 mm Petri plates containing PDA medium. For colony characters like colony diameter, colour, texture and pigmentation were studied and for spore dimensions length and width of both micro conidia and macro conidia and septation in macro conidia were recorded by following Ocular micrometry.
 
Aggressiveness
 
The pathogenicity of Fusarium isolates was done separately for their aggressiveness by following standard soil infestation method (Rubayet et al., 2017) in pot culture under the shade condition on three susceptible lentil cultivars viz. Maitri, HUL-57 and BM-6. The experiments were carried out in a greenhouse under controlled conditions (25°C and >80% humidity with continuous light). Ten seeds were sown in plastic pots (15 cm diameter) containing mixture of sterilized soil and vermicompost. The culture of each Fusarium isolate was prepared by inoculating them into maize kernels, then incubated at 25±2°C for 10 days, after which periodic observations were taken for the percentage of disease incidence for each isolate.
 
DNA isolation
 
50 mg mycelial mat from 7 days old PDB culture was collected by straining in filter paper and crushed with liquid nitrogen then 600 µl Buffer FG1 added to this powdered dried tissue vortex vigorously to mix. Incubated at 65 p C for 30 minute and aspirate 300 µl supernatant to a new 1.5 ml microfuge tube. Adding 150 µl Buffer FG 2 followed by 300 µl absolute ethanol and vortex. Add 400 μl of Buffer BL into the spin column, incubated at room temperature for 2 minutes, centrifuged at 12000 rpm for 2 minutes and discard the flow through. Transfer column to a second collection tube and wash by adding 650 µl DNA Wash Buffer diluted with absolute (96%-100%) ethanol. Centrifuge at 10,000 g for 1 minand discard the flow-through liquid. Transferred column to a clean 1.5 ml microfuge tube and 100 µl Elution Buffer added and the centrifuge tube for further use. DNA concentration was estimated using an UV vis spectrophotometer (Thermo Spectronic UV1). It was then stored at -20°C until further use (Datta et al., 2011).

Morphological variability
 
Single spore-cultures of 9Fusariumisolates which were collected from different locations (Fig 1) showed cultural variability in respect of morphology of mycelium, colony colour, texture and margin on PDA. Colour of the colony showed differences among the isolates on PDA media (Fig 2). Colony colour varied from white or pink-white and with time they changed to light brown colour with increased in the age of the fungal cultures within the media (Table 1). Colony diameter was found highest in case of Fusarium isolate W2 (8.87 cm) and minimum colony diameter was noticed in the isolate W3 (8.28 cm). Nine different isolates showed their variability in terms of their conidia size (Fig 3 and Table 2). The maximum average length of micro conidia was found in W2 (15.07±0.60 μm) and minimum length of micro conidia was noticed in W8 (12.69±1.03 μm. In case of breadth of micro conidia, the maximum breadth was noticed on W6 (3.52±0.35 μm) and minimum in W1 (2.85±0.07 μm) and the number of septation of micro conidia ranged from 0-2. The maximum length of macro conidia was observed in W7 (30.82±1.16 µm) and minimum in W1 (21.57±0.52 µm). In case of breadth of macro conidia, the maximum breadth was noticed on W8 (6.27±0.24 μm) and minimum in W5 (3.53±0.04ìm) with an average of 0-4 septa. The results of this study match with Belabid et al., (2004) who reported a single race of F. oxysporum f. sp. lentis in Algeria where macro and micro conidia showed great variation in terms of size, shape and septation.
 

 

 

 

 

 
Virulence (Pathogenicity) of the isolates
 
Pathogenicity test by Weideman and Wehner (1993) showed that there was a significant variation in disease severity on lentil variety Maitri with nine different isolates (Fig 4, Table 3). The isolate W7 produced highest PDI (93.33%) and minimum disease severity was noticed on W1 (75.00%). Among the nine isolates it was found that, the isolates W7 produced maximum PDI and was highly virulent isolate. According to disease severity the isolates were grouped in following descending order W7>W8>W2> W3>W5>W4=W6=W10>W1. Considering the disease severity of all the isolates, the maximum PDI producing (most virulent) isolate was W7 (sample collected from farmer’s field of Berhampore, Murshidabad).
 

 

       
There was a significant variation in disease severity among the nine isolates of Fusarium on lentil variety HUL-57 with a few exceptions. The isolate W1, W4, W7 showed maximum disease incidence (100%) and minimum disease severity was noticed on W5 (86.66%). Among the nine isolates it was found that, the isolates W1, W4, W7 produced maximum PDI and were highly virulent isolate. The degree of disease severity on the variety BM6,the isolate W1, W2, W3, W6, W7 and W10 showed maximum disease incidence (100.00%) whereas minimum disease severity was noticed on W4(93.10%). Though F. oxysporum f. sp. lentis has a narrow host range, it does exhibit great variability in virulence/aggressiveness as has been reported earlier (Mondal et al., 2020).
 
Molecular variability of different isolates of Fusarium sp.
 
Identification of molecular variability was performed by sequence analysis of rDNA-ITS region of each isolate for confirmation of molecular variability of the Fusarium isolates.
 
Molecular identification of Fusarium sp. based on ITS
 
The total size of the ITS1 and ITS4 regions, including the 5.8S rDNA gene of the isolates studied varied from 371 to 568 bp (Fig 5). The sequences were identified and deposited in NCBI GenBank, phylogenetic tree was also constructed using Neighbor-Joining (J) method of mathematical averages (UPGMA). W2, W3, W4, W6, W7, W8, W10 were identified as Fusarium oxysporum with the accession number MN758599, MN758600, W4 MN758601, MN758603, MN758604, MN758605, MN818591 respectively and W1 and W5 as Fusarium equiseti MN758598 and MN758602 respectively (Table 4).
 

 

 
Sequence analysis of ITS (Fusarium sp.) and evolutionary  relationship
 
ITS sequences of Fusarium isolates W1-W10 were aligned with the consensus region using CLUSTAL W program. Phylogenetic analysis grouped the Fusarium isolates into two clusters, Cluster I include Fusarium isolates W2, W3, W4, W6, W7, W8, W10 with a bootstrap support of 47% and cluster II includes Fusarium isolates W1 and W5 with a bootstrap support of 95% (Fig 6). Singha et al., (2016) has stated that ITS sequence analysis is one of the most accurate methods of revealing taxonomic and phyllogenetic relationships among Fusarium complex. Identity matrix of the Fusarium isolates ranged between 91.7% to 100%. Genetic identity of the isolates retrieved from NCBI Gen-bank obtained was 91.8% to 100% (Table 5). A report on the phylogenetic tree by Nirmaladevi et al., (2016) based on the ITS sequence analysis revealed four major groups suggesting four major evolutionary lineages of Fusarium oxysporum f. sp. lycopersici existing in tomato growing regions of India.
 



 

The present study reports wide distribution of lentil wilt disease in the lentil growing parts of West Bengal, India and showed prevalence of morphologically and genetically diverse isolates of Fusarium oxysporum possessing considerable pathogenic variability. Sequence analysis of rDNA-ITS region along with morphological and pathogenic data for characterization of the isolates greatly enhanced the understanding of the variability within this important fungus. This could ultimately benefit for the management of wilt disease through host plant resistance.

The authors are also grateful to Department of Plant Pathology, B.C.K.V, West Bengal for providing necessary support and facilities during the course of experiment.

All authors declare they have no conflict of interest with this article.