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

Background: Fusarium wilt in lentil ( Lens culinaris L.) infected by Fusarium oxysporum is considered as one of the major biotic factors for low productivity in lentil. The present study was conducted to determine nine Fusarium isolates (W1-W8 and W10) which were isolated from different lentil growing fields of West Bengal, India and were characterized for their physio-morphological, biochemical and molecular traits in vitro . Methods: Nine Fusarium infected lentil plant samples were collected from 9 different places of West Bengal. Pathogen was isolated from lentil samples using PDA media followed by cultural, morphological, pathogenic and molecular variability studies for all the isolates of Fusarium. Type of pigmentation, sporulation, conidia size of each isolate was recorded by observing culture plate after complete growth of the mycelium. Pathogenicity test were done to check the aggressiveness of all the 9 isolates of Fusarium in three varieties of lentil (Maitri, HUL-57 and BM-6). The isolated fungal DNA was amplified with specific primer pair, Forward primer ITS 1 and Reverse primer ITS 4. Result: Morphological assessment of 9 isolates showed distinct identification characters and variability. The mycelia of all the isolates were septate, profusely branched with differential pigmentation, texture and margin.All the Fusarium isolates produced micro-and macro-conidia in pure culture within seven days after inoculation and showed different morphological characters in pathogenicity test, variety Maitri showed better germination (100% in W2, W4, W6, W7, W8 and W10) and less disease severity against most of the strains (75% in W1) as compared to other two varieties against and hence variety Maitri taken into consideration for greenhouse and field trials. Furthermore, identification of molecular variability was performed by sequence analysis of rDNA-ITS region of each isolate and sequencing of rDNA-ITS region supported the morphological study and confirmed the associated fungi at species level.


INTRODUCTION
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 andCappelli, 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).

Data analysis
The data were analyzed statistically by following appropriate designs for each of the experiments.The wilt incidence in the pathogenicity test was analyzed by CRD test (Tukey, 1949) and the ranking was also given for each isolate.

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 W 8 (12.69±1.03μm.In case of breadth of micro conidia, the maximum breadth was noticed on W 6 (3.52±0.35μm) and minimum in W1 (2.85±0.07μm) and  Morpho-molecular and Pathogenic Variability of Wilt of Lentil from Indo-Gangetic Plains of India  Morpho-molecular and Pathogenic Variability of Wilt of Lentil from Indo-Gangetic Plains of India the number of septation of micro conidia ranged from 0-2.The maximum length of macro conidia was observed in W7 (30.82±1.16µm) and minimum in W1 (21.57±0.52 µm).In case of breadth of macro conidia, the maximum breadth was noticed on W 8 (6.27±0.24μm) and minimum in W 5 (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 W eideman and W ehner (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 W 7 produced highest PDI (93.33%) and minimum disease severity was noticed on W 1 (75.00%).Among the nine isolates it was found that, the isolates W 7 produced maximum PDI and was highly virulent isolate.According to disease severity the isolates w ere grou ped in fo llow in g descend in g order W 7>W 8>W 2> W 3>W 5>W 4=W6=W 10>W 1. Considering the disease severity of all the isolates, the maximum PDI prod ucing (m ost viru len t) isolate was W 7 (samp le co llec ted fro m farmer's field of B erh am po re, 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 W 1, W 4, W7 showed maximum disease incidence (100%) and minimum disease severity was noticed on W 5 (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 W 1, W 2, W3, W6, W 7 and W 10 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.
Morpho-molecular and Pathogenic Variability of Wilt of Lentil from Indo-Gangetic Plains of India Morpho-molecular and Pathogenic Variability of Wilt of Lentil from Indo-Gangetic Plains of India   The optimal tree with the sum of branch length = 99.32 is shown.All 9 Fusarium sp.isolates along reference isolates are indicated in the tree.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 W 1 and W 5 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.

CONCLUSION
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.

Fig 1 :
Fig 1: The map of areas where Fusarium oxysporum isolates were collected from Lentil growing areas of West Bengal State, India.

Fig 2 :
Fig 2: Fusarium sp.growth in PDA medium after 7 days of incubation.

Fig 6 :
Fig 6:The neighbor-joining tree of Fusarium isolates based on ITS region sequences.

Table 1 :
Cultural studies of Fusarium sp. on PDA media.

Table 2 :
Variability in conidial shape and size of Fusarium sp.isolates grown on PDA.

Table 3 :
Pathogenecity tests of different Fusarium isolates against three susceptible variety of lentil viz.MAITRI, HUL-57 and BM-6 under artificial growing condition.

Table 4 :
Identification of Fusarium isolates based on ITS sequences through BLASTN search of the GenBank database.

Table 5 :
Identity matrix of Fusarium isolates considered in this study with other Indian and World isolates based on ITS sequence analysis.