Cluster analysis
Cluster analysis of the 10 most virulent isolates of
Lasiodiplodia theobromae yielded two major clusters, I and II. The major cluster I had two subclusters, Ia and Ib while major cluster II had only one subcluster. Two isolates each from subcluster Ia and Ib and one isolate from major cluster II were selected. Cluster analysis has been depicted in Fig 1.
The subcluster Ia was divided into two groups; From the first group containing the isolates KoKBn and PVaT, KoKBn was selected being the only one isolate from Kottayam. PMuNe was selected from the second group being the only variant present in that group. The subcluster Ib had two groups; First group of I b was divided to two subgroups; PMuM from first subgroup was avoided as one isolate from Palakkad has been selected earlier. From second subgroup containing KPKa and KPB, KPB was selected based on earliness in pycnidia production which could be related to higher virulence. From the second group containing TMNe and WMnMa, WMnMa was selected due to variety specificity. From cluster II containing TNaM and WAmMa, TNaM was selected due to location specificity. Therefore, selected isolates were TNaM, WMnMa, KPB, PMuNe and KoKBn. Hence, these representative isolates were collected from five different districts and different varieties.
Evaluation of pathogenic variability of different isolates of the pathogen
All the five isolates caused 100 per cent incidence on Banaganapally variety. PDI ranged between 66.67-100. Among the isolates, isolate TNaM caused highest PDS of 73.3% on Neelam variety whereas PMuNe showed lowest PDS of 13.33% on Imam Pasand. Largest lesion (0.95 cm) was caused by KPB on Muvandan while smallest lesion (0.28 cm) was caused by PMuNe on Imam Pasand. KPB took minimum number of incubation days (9) on Muvandan while WMnMa and PMuNe took maximum number of incubation days (17.33) in Neelam and Imam Pasand respectively. Different symptoms caused by the isolates include black discolouration on inoculated area and cracking of outer cortex. Some isolates were able to cause drying of mango seedlings;
i.e TNaM, WMnMa, KPB and KoKBn caused drying of twigs in Neelam and KoKBn caused drying of twigs in Priyur. On Neelam variety, KPB caused drying of twigs 29 DAI (days after inoculation); WMnMa 34 DAI; KoKBn 37 DAI and TNaM caused drying 44 DAI. On Priyur variety, KoKBn caused drying of twigs 41 DAI. At the outset, lesions formed on the plants and progressively spread across the entire stem; subsequently leaves started showing wilting and yellowing symptoms. A week later, the leaves turned brown in colour and by the following week, leaves started falling. Experimental layout has been represented in Fig 2 and pathogenic variability evaluation of
L. theobromae isolates on different mango varieties has been represented in Fig 3i and 3ii.
Variations were discerned significantly different among the isolates for different pathogenic parameters. PDI, PDS, size of lesion, days taken for symptom development (incubation days/latent period), virulence index for different isolates, CI value for different varieties and categorization of mango varieties based on CI value have been furnished in Table 4, 5, 6, 7, 8, 9 and 10
respectively.
The isolate TNaM produced substantial variation for different pathogenic parameters like PDI, PDS, lesion size and incubation days on different mango varieties. Range of PDI was 66.67-100 per cent with maximum and minimum disease incidence of 100 per cent on Bennet Alphonso, Neelam and Banganapally and 66.67 per cent on Priyur, Muvandan and Imam Pasand respectively. PDS ranged between 26.64-73.33 per cent with highest of 73.33 per cent on Neelam and lowest of 26.64 per cent on Priyur and Imam Pasand. Lesion length ranged between 0.4-0.76 cm with largest and smallest lesion on Banganapally (0.76 cm) and Priyur (0.4 cm) respectively. Incubation days ranged between 11.67-16 with minimum of 11.67 days on Bennet Alphonso and maximum of 16 days on Priyur and Imam Pasand.
Appreciable variation was discerned for different pathogenic traits like PDI, PDS, incubation days and lesion size by the isolate PMuNe. Range of PDI was 33.33-100 per cent with highest and lowest incidence of 100 per cent on Bennet Alphonso, Imam Pasand, Banganapally and Priyur and 33.33 per cent on Muvandan respectively. PDS ranged between 13.33-53.28 per cent with maximum on Bennet Alphonso and Banganapally (53.28%) and minimum on Imam Pasand (13.33%). Lesion length ranged between 0.28-0.88 cm with largest and smallest lesion on Bennet Alphonso (0.88 cm) and Imam Pasand (0.28 cm) respectively. Range of latent period was 10-17.33 with minimum and maximum days of 10 on Bennet Alphonso and 17.33 on Imam Pasand respectively.
On different mango varieties, the isolate, WMnMa displayed considerable diversity variation for different pathogenic attributes like PDI, PDS, incubation days and lesion size. PDI ranged between 66.67-100 per cent. Cent per cent disease incidence was exhibited by WMnMa on Neelam, Imam Pasand and Banganapally and lowest incidence (66.67%) on Priyur, Muvandan and Bennet Alphonso. Range of PDS was 26.64-66.67 per cent with highest and lowest on Neelam (66.67%) and Bennet Alphonso (26.64%) respectively. Lesion length ranged between 0.41-0.78 cm with largest and smallest lesion on Banganapally (0.78 cm) and Priyur (0.41cm) respectively. Incubation days ranged between 10.33-17.33 with minimum and maximum of 10.33 on Banganapally and 17.33 on Priyur respectively.
Clear variation was detected for different pathogenic parameters like PDI, PDS, incubation days and lesion size by the isolate, KPB. PDI ranged between 66.67-100 per cent with maximum and minimum incidence on Priyur, Muvandan, Neelam and Banganapally (100%) and Imam Pasand and Bennet Alphonso (66.67%) respectively. PDS ranged between 26.64-70 per cent with highest and lowest on Neelam (70%) and Imam Pasand (26.64%) respectively. Range of lesion length was 0.56-0.95 cm with maximum and minimum lesion size on Muvandan (0.95 cm) and Bennet Alphonso (0.56 cm) respectively. Latent period ranged between 9-16 with minimum and maximum number of days on Muvandan (9) and Imam Pasand (16) respectively.
The isolate KoKBn exhibited evident variation for different virulence traits like PDI, PDS, incubation days and lesion size on different mango varieties. PDI ranged between 66.67-100 per cent with maximum disease incidence (100%) on Priyur, Imam Pasand, Bennet Alphonso and Banganapally and minimum incidence (66.67%) on Muvandan and Neelam. Range of PDS was 33.3-66.67 per cent with maximum and minimum on Priyur (66.67%) and Muvandan (33.3%) respectively. Lesion length ranged between 0.31-0.9 cm with largest and smallest lesion on Bennet Alphonso (0.9 cm) and Neelam (0.31 cm) respectively. KoKBn took minimum number of days for symptom initiation of 10 on Bennet Alphonso and maximum number of days of 17.33 on Neelam and incubation days ranged between 10-17.33.
Different works have been done on pathogenic variability of different pathogenic fungi while the pathogenic variability of
Lasiodiplodia theobromae has not been investigated yet. The variation observed in the current study is in congruence with the variation observed by
Vitthal (2015) who studied pathogenic variability among 12 isolates of
Colletotrichum capsici responsible for leaf spot of turmeric. According to his work, incubation days and number of spots ranged from 7 to 13 and 3 to 6 respectively. Range of disease incidence and severity was 35-66 per cent and 30.51-38.32 per cent respectively.
Ahmad (2016) identified the isolate Kti 10 with average disease intensity of 10.3 per cent as most virulent among 12 isolates of
Exserohilum turcicum when artificially inoculated on 12 maize genotypes. In another experiment, he studied variability of these 12 isolates on another maize cultivar, Pahalgam local cultivar wherein range of different virulence traits like incubation days, PDI, breadth and length of lesion was 4.07-7.74, 20.23-30.5 per cent, 0.48-0.95 cm and 2.88-8.42 cm respectively.
Lakshmi Prasad et al. (2020) categorized 220 isolates of
Alternaria helianthi into three different groups
viz., low, medium and high aggressive groups based on disease reaction on six sunflower accessions. More number of isolates fell under medium aggressive group.
The pathogenic variability reported in this study in also in consonance with the variation observed by
Mohammed et al. (2022) in isolates of
Alternaria porri collected from different parts of Uganda which produced PDS ranging between 16.7-56.3% and the onion variety Red coach was found to be resistant.
Das et al., (2023) also observed variation in nine isolates of
Fusarium oxysporum in three wilt susceptible cultivars of lentil wherein PDI ranged between 75-100% and isolate W7 was found to be most aggressive. Pathogenic variability was evaluated for ten isolates of
Alternaria cyamopsidis causing
Alternaria blight in clusterbean whose disease intensity ranged between 34.69-65.50%. The most and least virulent isolate was AlcyJp1 (disease intensity of 65.50%) and AlcyBk2 (disease intensity of 34.69%) respectively
(Sharma et al., 2024). Pathogenic variability was evaluated based on size of necrotic lesion produced by
Macrophomina phaseolina by
Amrate et al., (2024). They concluded that the isolates Mp-1 to be most virulent (15.3 cm) and Mp-8, Mp-10 and Mp-12 to be least virulent (3 mm). Similar results were obtained in the area under the lesion progression curve also.
Errampalli et al. (2024) categorized six isolates as highly virulent with >50% average mean over all the cultivars among 89 isolates of
Fusarium oxysporum f. sp. ricini collected from 11 Indian states.
Jojy et al. (2024) also noticed diversity in isolates of
Colletotrichum capsici in pathogenic characters like incubation days, lesion size, and PDI which ranged between 1-3, 0.17-1.13 cm and 16.67-45.33% respectively.
The variation in symptom expression of different isolates on different varieties may be due to variation in climatic conditions or variation in inherent resistance of different varieties or variation in genotype of isolates. Genetic variation of
Colletotrichum isolates inciting mango anthracnose
(Bincader et al., 2022) and
Alternaria isolates causing cotton leafspot (
Sampathkumar and Raghavendra, 2024) was established using SSR primers and ISSR-PCR respectively. Variation may arise from interaction of above-mentioned factors also. Since India is one of the centres of origin of mango, it could be the centre of diversity of its pathogen
Lasiodiplodia theobromae also. This can lead to pathogen variability because diversity of
Lasiodiplodia theobromae has been larger in areas where natural host population exist than the regions where the host has been introduced.
Variability in phytopathogens is driven by various phenomena such as host specialization, selection, mutation, recombination
etc. Strong directional selection intensifies when a resistant gene becomes common in an area. This favours the increase in virulent mutant so that break down of resistance happens. The extent to which selection occurs is determined by degree of host specialisation resulting in increased variations in pathogen population.
Ability of
Lasiodiplodia theobromae isolates collected from dragon fruit to infect other fruit crop such as mango, guava and banana
(Briste et al., 2022) indicates that the fungus can infect multiple host species. Under natural conditions, this can lead to the change of pathogenicity level, contributing to pathogenic variability. Mango intercropping and mango trade across the state Kerala introduces diverse genotypes of the pathogen. Additionally, intercropping facilitates maintenance and continuous spread of distinct pathogen strains every season.
In the current investigation, the mean virulence index was found highest for the isolates KoKBn (4.19), KPB (4.08) and TNaM (3.35). Thus, these isolates were the most virulent among the five isolates. Range of virulence index was 2.78-4.19. This finding is in harmony with
Vaniya et al. (2022) who reported that range of virulence index was 2.18-10 in her studies on variability of
Sclerotium rolfsii isolates inducing stem rot of Indian bean.
Varietal screening for dieback resistance
In the present study, lowest CI was observed for Imam Pasand and highest for Banganapally. Range of CI was 27.08-50.61. This finding is in line with
Sangeetha (2008) who screened thirteen mango varieties to dieback disease and reported that the range of CI was 0.63-31.88.
In the present investigation, based on CI value the varieties Neelam, Bennet Alphonso and Banganapally were categorized as susceptible (S) to mango dieback whereas the varieties Priyur, Muvandan and Imam Pasand were categorized as moderately susceptible (MS) to mango dieback (Table 10). This corroborates the reports by
Jain and Depale (2023) who observed that maximum lesion size (69.99 cm
2) was produced in Neelam variety and it was categorized as susceptible to stem end rot of mango caused by
Botryodiplodia theobromae. Findings in our present work is in slight conformity with
Khanzada et al. (2015) who reported that the mango variety Neelam was showing moderate susceptibility to
Lasiodiplodia theobromae inciting mango decline. In the current study, all the isolates produced 100 per cent incidence on the variety Banganapally, which proves its susceptible nature.
Differential response of different mango cultivars to mango dieback may also be related to variation in amount of different metabolites of the pathogen like defense related enzymes, antifungal compounds, phenols, pectin
etc which has to be studied in detail. Different compounds like lasiodiplodin and jasmonic acid
(Salvatore et al., 2020), virulence factor of the pathogen like NmrA-like proteins
(Peng et al., 2022) have been isolated from
Lasiodiplodia theobromae. Different varieties may be having varied amount of above mentioned compounds which might be the reasons for varying resistance and this aspect also has to be investigated in future research. According to some reports, the resistance of different mango varieties to dieback disease was related to presence of chitinase enzyme. According to
Karunanayake et al., (2014) elevated proportion of chitinase in mango cultivars Rata and Kohu contributed resistance to the infection caused by
Lasiodiplodia theobromae inciting stem end rot in mango. Different scientists have documented differences in the accumulation of defense molecules in various cultivars. Varying level of phenol (
Alañón et al., 2021;
Mandal et al., 2021), terpenoids (
Shimizu et al., 2021), phenol and antioxidant capacity
(Troiani et al., 2022) and terpenoids and phenols
(Tandel et al., 2023) have been reported in different mango cultivars. This variation in different compounds may be accounted for differential response of mango germplasm to mango dieback disease, necessitating further research. The differential reactions of the germplasm might be important to manage mango dieback through gene deployment.