Distribution and diversity of Fusarium oxysporum f.sp. cubense TR4 causing banana wilt in Sub-tropics of India and comparative analysis of TR4 specific molecular detection methods

Fusarium wilt caused by Fusarium oxysporum f.sp. cubense (Foc) is regarded as the most recurring and deadliest threat to banana cultivation worldwide. Among three physiological races of the pathogen capable of infecting Musa spp., Tropical race 4 (TR4) is considered the most virulent race of Foc without any known source for resistance. In India, race 1 was commonly observed to affect the cultivars of Pisang Awak, Rasthali (Silk), and Malbhog (Silk) while the Grand Naine (G9) the most popular and extensively grown cultivar of Cavendish banana in subtropics of Uttar Pradesh (U.P.) and Bihar was free from the disease till 2018. But in 2018, the incidence of wilt was first time observed in Sohawal, Ayodhya (U.P.) later in Katihar (Bihar) of India. The Association of TR4 was later confirmed with diseased samples. In the current study, the distribution and incidence of TR4 was assessed in the subtropics covering the states of U.P. and Bihar. Forty-one isolates pathogenic to the banana plant were collected from 6 states, which varied in terms of aggressiveness, and morphological characteristics like colony color, spore size, and shape. The isolates from U.P. and Bihar were characterized as TR4 using two primer sets targeting the SIX1a (secreted in the xylem) gene however isolates from Gujarat, Assam, Andhra Pradesh, and Tamil Nadu (TN) were identified as Race 1 using two primer sets targeting SIX6b and a hypothetical protein (XM_018394505.1). Genetic variability was assessed using 8 ISSR markers and divided all 41 isolates into two major groups, with a set of isolates from the U.P. and Bihar in group A and isolates from other states in group B. In the comparative analysis of three TR4-specific primers, only 1 out of 3 primer sets was confirmed to be more accurate in the characterization of the TR4. Analysis of molecular variance revealed that genetic variations among the regions and within the region accounted for 37.0 per cent and 63.0 per cent. The current study signified that the spread of the TR4 in U.P. and Bihar suggests the need for efforts for management with emphasis on biosecurity practices and the use of antagonistic biological agents to prevent further spread.


Introduction
Worldwide bananas and plantains (Musa spp.) are the most widely produced, traded in terms of export value, and consumed fruit among different fruits. It is cultivated across 135 countries across tropical as well as subtropical environmental conditions (Magdama et al. 2020). Notably, the Cavendish type banana is the ruling variety which is being popularly cultivated and traded at the global level accounting for 47 per cent (50 billion tonnes annually) of worldwide production (mentioned in https:// www. fao. org/ econo mic/ est/ est-commo dities/ oilcr ops/ banan as/ banan afacts as accessed on 11-02-2023). Fusarium wilt, a soil-borne disease also termed as Panama wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is a recurring threat to global banana cultivation. Foc infection starts from the banana roots and spreads to the vascular region. The infection blocks the plant's vascular system, thereby causing wilting and eventual death (Bai et al. 2020). The external symptoms of Panama wilt include yellowing and necrosis of older leaves, pseudostem splitting at the base, and reddish to dark brown discoloration of the vascular system. The dried leaves collapse at the petiole giving the typical skirt-like appearance and eventually the entire plant dies (Dita et al. 2018). The fungus reproduces asexually with an ability to survive as chlamydospores in infested banana fields for up to 3 decades which means once the field soil gets infested with Foc, susceptible banana cultivars cannot be raised successfully in the same field for at least 30 years.
Foc is a highly diverse pathogen and based on its pathogenicity to different host cultivars Foc has been classified into three evolutionary physiological races, with Foc race 1 (R1) affecting 'Gros Michel' (AAA), 'Lady Finger' (AAB) and 'Pisang Awak' (ABB) bananas; Foc race 2 (R2) affecting 'Bluggoe' (ABB) and closely related ABB clones; and Foc race 4 (R4) affecting the 'Cavendish' banana subgroup (AAA) (Siamak and Zheng 2018). Foc R1 gained notoriety in mid twentieth century when an epidemic occurred after the extensive monoculture of popular 'Gros Michel' cultivar which was susceptible to R1 and this epidemic threatened the entire banana industry then (Dita et al. 2018). Eventually, the susceptible 'Gros Michel' was replaced by Cavendish cultivars which has resistance to Race 1 during the 1960s. The resistance in Cavendish cultivars was broken by tropical race 4 of Foc (Foc TR4) which originated in Indonesia (Ordonez et al. 2015;Zheng et al. 2018). In addition to physiological races, the diversity of Foc can be evaluated through the assessment of vegetative compatibility among isolates. Isolates that belong to the same vegetative compatibility group (VCG) possess identical alleles at vic loci, while isolates from different VCGs differ in alleles at vic loci (Mostert et al. 2017). Presently, 24 VCGs have been identified for the banana wilt pathogen, but cross-compatibility among VCGs also exists thus forming VCG complexes (Fourie et al. 2009). However, the VCG assay is a timeconsuming and labor-intensive technique. Moreover, vegetative compatibility is unable to identify the degree of relatedness or distinctness among the vegetative compatible or incompatible groups (Karangwa et al. 2018). The approaches to assess the genetic diversity and evolutionary relationship among Foc isolates have been advanced in the past 2 decades and different molecular markers viz., random amplified polymorphic DNA (RAPD) markers, restriction fragment length polymorphisms (RFLP), amplified fragment length polymorphism analyses (AFLP), inter simple sequence repeats (ISSRs), simple-sequence repeats (SSRs) markers have been employed (Bentley et al. 1995;Groenewald et al. 2006;Thangavelu et al. 2012;Maryani et al. 2019).
India with a 21 per cent share in global banana production is also facing the threat of the devastating Fusarium wilt disease (Damodaran et al. 2018;Thangavelu et al. 2019). Foc TR4 was detected in India from Sohawal, Uttar Pradesh (UP), and Katiyar, Bihar in 2018 (Damodaran et al. 2018;Thangavelu et al. 2019). Since then, it has been spreading over larger areas of the Indian sub-tropics (U.P. and Bihar) accounting for severe economic losses and a decline in banana production (Damodaran et al. 2020). Cavendish bananas constitute a major share of the cultivars being grown in the Indian subcontinent. The states like Tamil Nadu (TN), Karnataka, Andhra Pradesh (AP), and Assam are known for the indigenous diversity of traditional banana cultivars. Among them, the popular varieties of the regions like Malbhog in Assam, Rasthali in TN, and Amritpani in AP that comes under the Silk group of cultivars were known to be highly susceptible to Foc Race1 that results in a gradual decline in the areas under cultivation (Thangavelu et al. 2012). The cultivar Pisang Awak predominantly grown as Karpooravalli in Southern India is also reported for its susceptibility to the Foc Race 1 (Kavino et al. 2008;Damodaran et al. 2009).
The pathogen disseminates to new areas with the transportation of asymptomatic planting materials, relocation of farm implements and soil adhered to farm equipment, shoes of workers, vehicles, etc. from infested fields to noninfested ones (FAO 2020). Since the introduction of TR4 in India, the data on the prevalence of Foc TR4 in the subtropics and also information on the status of its incidence in the other traditional cultivars that are susceptible to Foc R1 are limited. Moreover, it is necessary to identify the hotspots of TR4 and the most specific race-specific primers to develop a national strategy for the management and prevent the dissemination of the pathogen to newer areas. The major objectives of the current study were to (a) assess the genetic diversity among Foc isolates using molecular markers (b) validation of available TR4-specific primers, and (c) assess the race status of the Foc present in susceptible dessert cultivars of Assam, TN, Gujarat, and AP using race-specific primers.

Survey and collection of diseased samples
Transect surveys were conducted in different districts of U.P., and Bihar, for assessing the status of banana wilt in these states. In U.P., 22 districts viz. Pilbhit, Kheri, Sitapur, Lucknow, Barabanki, Raebareli, Fatehpur, Kaushambi, Bahraich, Gonda, Faizabad, Amethi, Pratapgarh, Allahabad, Ambedkar Nagar, Basti, Sant Kabir Nagar, Siddhartnagar, Gorakhpur, Maharajganj, Kushi Nagar, and Deoria were surveyed. In Bihar, Sitamarhi, Vaishali, Bhagalpur, Purnia, and Katihar districts were surveyed for assessing the incidence of wilt. Plants were observed for the disease symptoms peculiar to wilt. To confirm the Fusarium wilt incidence, a vertical section of pseudostem was cut and observed for the presence of any ooze or foul smell. The plants which were severely infected were horizontally cut, and the pattern of internal discoloration of pseudostem (whether concentrated to the periphery or center) was observed. The number of diseased plants in fields was noted (I) and percent disease incidence was calculated as DI (%) = (I/T)*100 where T is the total number of plants in the field (information given by farmers of respective fields).
Diseased samples were collected by cutting a longitudinal section of the pseudostem of the banana plant with typical yellow leaf syndrome, pseudostem splitting, and internal red discoloration of vascular bundles. The samples were properly packed in paper bags, labeled, and brought to the laboratory for isolation. Additionally, surveys were also conducted in Goalpara district of Assam, Surat district of Gujarat, Coimbatore district, Horticultural Research Station (HRS), Thadiyankudisai of TN, and HRS, Dr, YSR Horticultural University, Kovvur, AP, and isolates were collected. The sampling locations along with their GPS coordinates have been mentioned in Table 1.

Isolation, morphological characterization, and maintenance of isolates
The discolored vascular strands were pulled out from the samples using a flame-sterilized scalpel blade and forceps. The vessels were left for natural drying by placing them between the sterilized filter papers for one or two days. After the drying of the vessels, small sections of these dried vessels (2-5 mm) were cut and placed on quarterstrength potato dextrose media (1/4 PDA) supplemented with chloramphenicol antibiotic. The fungal colonies morphologically similar to Fusarium spp. were sub-cultured on PDA supplemented with antibiotics. Pure cultures of each fungal isolate were developed through the single spore method following the procedure previously described (Ho and Ko 1997) with slight modification. Briefly, diluted spore suspension was plated on 2 per cent water agar followed by incubation for 12 h at 25 °C ± 1. The germinating single spores were marked under the microscope and transferred to fresh PDA plates under aseptic conditions. The plates were incubated at 25 °C ± 1. The colony characters, size, and shape of micro as well as macroconidia were recorded as described by Leslie and Summerell (2006). For observing the size of micro-, macroconidia, and chlamydospores, the isolates were also inoculated on synthetic nutrient-poor agar (SNA) (comprised of 1 g of KH2PO4, KNO3; 0.5 g of MgSO4·7H2O, KCl; 0.2 g of Glucose and Saccharose, and 20 g of Agar in 1 L of distilled water) and incubated at 25ºC + 1. Microscopic observations were taken on the 23rd day of inoculation.

Pathogenicity assays of the isolates
The pure cultures maintained on PDA were used for the pathogenicity assay of the isolates. At least one isolate from a district was selected for pathogenicity assay, however, more than one isolate/district was included in case they were morphologically dissimilar. Thirteen isolates viz., FKB-1, FKB-4, FKB-6, FKB-8, FKB-15, FSoUP-1, FSkUP-3, FMUP-2, FAbUP-4, FCoTN-1, FKoAP-1, FSuGuj-1, and FGuAS-1 were used to inoculate 3 months old banana plants cv. Grand Naine (G9) keeping 3 replications for each isolate. Three months old secondary hardened tissue cultured banana plantlets cv. Grand Naine were used for pathogenicity assays. The spore suspension was prepared using 10-15 days old culture in autoclaved distilled water under aseptic conditions. The number of spores was adjusted to 106 to 108 conidia/ml using hemocytometer. The inoculations were carried out by dipping the plants with trimmed roots in spore suspension of respective isolates for 30 min as described by Perez-Vicente and Dita (2014). Control plants were dipped in sterilized water and then planted. The plants were then transplanted in pots (30 cm) containing sterilized clayey loam soil (pH ~ 7.0) and pots were kept in the greenhouse where the temperature ranged from 29 to 34•C with 60-70% humidity during the experimental period. The plants were observed for symptoms development. After 60 days the plants were uprooted and their pseudostem and rhizome were observed for discoloration development. Discolored rhizomes were used for the re-isolation as described in the previous section. Data on leaf symptoms index and vascular discoloration were recorded based on the rating scale developed by the International Network for the Improvement of Banana and Plantains (INIBAP) (Carlier et al. 2002).

Identification of races and comparative analysis of TR4-specific primers
After confirming the pathogenicity of the isolates, the race of 41 isolates has been identified using the primers developed by Thangavelu et al. (2022). The fungal DNA was extracted using HiPurA™ Fungal DNA Purification Kit (Himedia) following the manufacturer's instructions. The PCR was performed using primers (Table S1) and protocol as described by Thangavelu et al. (2022). Additionally, the efficacy and specificity of 3 other TR4specific primers developed by previous workers (Dita et al. 2010;Li et al. 2013;Carvalhais et al. 2019) were also assessed following their thermal conditions in PCR (Table S1). The PCR experiments were performed at least twice.

Genetic variability analysis using ISSR markers
For genetic variability analysis, the first polymorphic primers out of 34 ISSR primers were identified by performing PCR using the DNA template of two Foc isolates FKB-1 (isolated from the cultivar G9 from the Katihar district of Bihar where TR4 was earlier reported) and FCoTN-1 (isolated from the Coimbatore district of TN from the cultivar Karpooravalli where Race1 was predominant). The PCR was performed in a final volume of 20 µl containing 2.0 µl of 50 ng DNA template, 1 µl of primer (10 picomol/ul), 2X master mix (EmeraldAmp® GT PCR Master Mix), and nuclease-free water to make the final volume. The amplification was performed in Himedia Prima-96™ Thermal Cycler using PCR conditions that included initial denaturation for 5 min at 94 °C followed by 35 cycles of denaturation at 94 °C for 30 s, annealing for 45 s and extension for 1.30 min at 72 °C followed by a final extension for 7 min at 72 °C. The PCR products were electrophoresed in 1.5 per cent agarose gel stained with ethidium bromide (0.5 µl/ml), bands were scored and the presence or absence of bands indicated as 1 or 0, respectively. A dendrogram was constructed using the NTSYS software based on the Jaccard coefficient through UPGMA agglomerative (Rohlf 1998). GenAlEx 6.5 (Peakall and Smouse 2012) a popular tool that works as an ad-ins in microsoft excel was used for population genetic analysis to generate AMOVA results of the FOC isolates. The analysis was carried out using the following parameter i.e., the organism as haploid and with 999 permutations. Nm is estimated as For population structure analysis, STRU CTU RE 2.1 software was utilized which runs on a Bayesian model method (Pritchard et al. 2000). Allele frequencies in each of the K populations and the degree of admixture A Monte Carlo Markov chain method were used to estimate each individual isolate. The analysis was performed using an admixture model with 10 independent simultaneous runs and 10,000 replications assuming K value ranging from 1 to 10. The best-fitted K was identified using the web-based program STRU CTU RE HARVESTER (Earl and Vonholdt 2012).

Symptomatology and disease incidence
In Bihar and U.P., the widely cultivated banana cultivar is Grand Naine or G9. In TN, Rasthali (Silk) and Karpooravalli (Pisang Awak) were mostly preferred along with other regional varieties. In Assam, Malbhog (Silk) is popularly cultivated, while in Gujarat, Grand Naine (G9) is a more popular one, and cultivated over an extensive area.
During the surveys and sample collection typical symptoms of Panama wilt were observed which included yellowing from the margins and eventually browning of older leaves, typical skirt-like appearance around the pseudostem due to the breakage of wilted leaves from petiole, and pseudostem splitting at the base. Upon cutting the pseudostem horizontally, characteristic reddish-brown discoloration was noticed. Additionally, there was the absence of any bacterial ooze or foul smell, thus nearly confirming the Fusarium wilt infection. The splitting of the pseudostem from the base and the typical ring or arc-type discoloration of the vascular region of the pseudostem near the base was the most prominent symptom of the banana Fusarium wilt. The disease incidence in U.P. state ranged from 10 per cent in Ambedkar Nagar to 60 per cent in Maharajganj district. Among 22 surveyed districts, the disease was observed in Pilibhit, Kheri, Barabanki, Ayodhya, Basti, Sant Kabir Nagar, Siddhartnagar, Mahrajganj, Gorakhpur, Kushi Nagar, Ambedkar Nagar, and Deoria. Four districts (Ayodhya, Maharajganj, Sant Kabir Nagar, Gorakhpur) were highly affected showing disease incidence spanning from 35-60 per cent. The Sohawal block of Ayodhya, Medhawal block of Sant Kabir Nagar, Captainganj block of Gorakhpur, and Siswa block of Maharajganj had a wide spread of the disease in an extended manner with high disease incidence percentage ranging from 30 to 60 per cent (Fig. 1a). In Bihar, transect survey was made from Katihar to Sitamadi covering the banana growing districts of the region. There was a severe incidence in the range of 30 to 65 per cent extensively in Katihar, Purnia, Baghalpur, and Vaishali districts. While extensive and moderate disease incidence of 20 per cent was observed in Sitamadi, and Bhagalpur districts (Fig. 1b). The locations of isolates included in the current study along with their GPS coordinates and disease incidence is given in Table 1. A total of 41 isolates from six states viz., Bihar, U.P., Gujarat, TN, A.P., and Assam were isolated on quarter strength PDA and further purified through single spore culture and maintained.

Isolation and morphological characterization of Fusarium oxysporum f.sp. cubense isolates
Upon isolation from the discolored vascular strand on quarter strength PDA, only growth of Fusarium oxysporum appeared without any contamination. The isolates produced white to light pink to purple pigmented mycelium. Most of the isolates produced aerial, cottony, circular mycelium. The isolates FKB-6 and FKB-13 transformed phenotypically characterized with flat wet white mycelium after 1-2 sub-culturing on PDA plates. On PDA, all the isolates produced abundant microconidia except FKB-3, FPTN-1, and 2. Micro-conidia were observed to be scattered singly and false heads were rarely observed. Most of the isolates produced ellipsoid-shaped single-celled microconidia however, FSuGuj-1, 2, and FPTN 1 and FPTN-2 produced microconidia with one septum. All the isolates except FPTN-1 and 2 produced abundant macroconidia. These macroconidia were straight to sickle-shaped with a round to tapering ends having usually 2-3 septation. Macroconidia produced by isolates FMUP-1 and 2 had 5-6 septations. Chlamydospores were also not produced by all the isolates on PDA even after the production of macroconidia. The chlamydospores produced were characterized by a smooth protective wall, terminally or intercalary formed mostly singly or in pairs. None of these isolates produced sporodochium. The morphological characteristics of all 41 Foc isolates are presented in Table 2.
Spore characteristics were also studied on SNA media (Table 3). All the isolates produced abundant micro-and macroconidia with varying sizes. Significant variation was obtained in the case of micro-, macroconidia, and chlamydospore size. Microconidia were formed in chains or false heads. Microconidia were oval to elliptical, with no septation, however infrequent occurrences of single septate microconidia were also observed. The longest microconidia were observed in the case of FSoUP-3 with 13.82-16.96 X 4.42-6.07 µm size (Length X Width) while isolates FCoTN-1a and 2 produced the smallest microconidia with 7.32-8.11X2.9-3.31 µm size. Conidia were formed in chains or false heads. Macroconida were straight to sickle-shaped with a round to tapering ends having 2-6 septations. Round, smooth-walled chlamydospores were formed in all the isolates except FMUP1, 2 and FPTN 1, 2.

Pathogenecity of Foc isolates
In the pathogenicity assay, all the isolates used for inoculation were pathogenic and produced typical symptoms of Panama wilt (Fig. S1a). All the isolates produced leaf symptoms characterized by streaking and yellowing of leaves in 28-45 days. Some isolates viz., FGuAs-1 and FSuGuj-1 did not manifest pronounced leaf symptoms except for slight yellowing of one older leaf from margins; although, vascular discoloration was quite evident in the case of all the isolates upon destructive sampling (Fig.S1b). Considerably, the isolates viz., FKB-1, FKB-8, FSkUP-3, and FMUP-2 with the highest leaf symptoms index (7) also had the highest vascular discoloration rating (7 & 9). Foc colonies were obtained upon re-isolation on quarter-strength PDA, which were morphologically and microscopically similar to the respective isolates. All the control plants remained free from any symptoms. The leaf symptom index and vascular discoloration rating (mean of replications) for the isolates are presented in Table 4. However, we did not find any trend between the virulence of isolates with geographical locations and host cultivars from which they were recovered.

Identification of races and comparative analysis of TR4-specific primers
Firstly, the races of 41 isolates were identified using primer sets developed by Thangavelu et al. (2022). The NRCB-TR4F & R primer set developed by Thangavelu et al. (2022) produced amplicons of ~ 250 bp specific for TR4 in the isolates from U.P. and Bihar but not in the isolates collected from Assam, Gujarat, TN, and AP (Fig. 2a). However, Fig. 1 Distribution of the Foc TR 4 in UP (A) and Bihar (B). The red marked districts indicate the locations with disease incidence more than 30 per cent NRCB-R1F & R produced the amplification product of size ~ 320 bp specific for Race 1 in the isolates collected from Gujarat, Assam, TN, and A.P. Therefore, the isolates collected from U.P. and Bihar were identified as TR4 while all the isolates from Gujarat, Assam, TN, and A.P. were designated as Race 1 (Fig. 2b). None of the isolates produced amplification of target size (~ 250 bp) with STR4 specific primers.
In the comparative analysis of TR4-specific primers, we observed that the primer pair (FocTR4-F/ FocTR4-R) developed by Dita et al. (2010) was unable to differentiate between the race 1 and TR4 and produced an amplification band of ~ 463 bp in all the fungal isolates including the ones isolated from the cultivars Virupakshi (Pome) and Amritpani (Silk) from Coimbatore and HRS, Thadiyankudisai, TN and HRS, Dr. YSR Horticultural University, Kovvur, A.P. (Fig. S2a). Likewise, the primer pair-W2987-F/W2987R failed to detect some of the TR4 isolates collected from Katihar, Bihar (FKB-6); Sohawal (FSoUP-1) and Ambedkar Nagar (FAbUP-1 and 3), U.P. However, no false positives were obtained in the case of Race 1 isolates (Fig. S2b). With primer pairs targeting the secreted-in-xylem genes of Foc (TR4F and TR4R) all the isolates of U.P. and Bihar were identified as TR4 as they produced amplification of size ~ 266 bp though this band was absent in the case of the isolates of Coimbatore and HRS, Thadiyankudisai, TN;  S2c). Hence based on our comparative analysis, 2 out of 3 primer pairs were inefficient and inaccurate in identifying TR4.
FGuAs-1 40 3 5 ISSR-34 were polymorphic (Table 5). A total of 77 fragments were amplified ranging from 0.2 kb-5 kb and a total of 34 bands were scorable. The maximum polymorphism was exhibited by ISSR-14 (AC)8G) with 71.42 per cent polymorphism followed by ISSR-10 (GGA(GAG)2ACG AGA) and ISSR-32 (CAG)5) with 55.55 per cent polymorphism. The dendrogram generated using the NTSYS software based on the Jaccard coefficient through UPGMA agglomerative showed diversity in the test pathogen population (Fig. 3). Based on the data generated from 8 ISSR markers, at 45 per cent similarity coefficient all the isolates identified as TR4 from U.P. and Bihar were clustered in Group A while isolates from TN, A.P., Gujarat and Assam identified as Race 1 were clustered in Group B. At 60 per cent similarity coefficient, group A was further divided into 5 subclusters. Cluster A1 was comprised of isolates FKB-1, 2, 3, 4, 9, 12, 14, 15; FMUP-1; FAbUP-2, 3, 4, 5, 6; FSoUP-1, 3 and FSkUP-1, 2, 3. Cluster A2 contained isolates FKB-5, 6, 8, 10, 11, and FMUP-2. Isolates FKB-7, FKB-13, and FAbUP-1 were placed in separate clades within Group A. In Group B, 5 sub-clusters were formed at 80 per cent similarity coefficient. In cluster B1, isolates from TN viz., FPTH-1, 2 and FCoTN-1, 2 along with 2 isolates from A.P. viz., FKoAP-1 and 2 were clustered together. Isolates FKoAP-3 and FKoAP-4 were clustered separately in single clades, respectively. Isolates from Gujarat (FSuGuj-1 and 2) and Assam (FuGuAs-1 and 2) were grouped in B2 and B5 clusters, respectively. Considerable low variability was found in the isolates collected from A.P., TN, Assam, and Gujarat where the similarity coefficient varied from 67 to 97 per cent. The genetic similarity among the isolates from Bihar and U.P. ranged between 46 and 100 per cent, and thus the isolates were highly variable. The genotyping of the 41 isolates from different locations using ISSR markers with uniform distribution in the genome revealed the existence of a significant difference between regions (P < 0.001) and among individuals within a population (P < 0.001) though the differences among the population were non-significant (P > 0.05) ( Table 6). The contribution of regional diversity and variation among individuals within the population to the total variation was 37 and 63 percent, respectively. The variation among the populations was non-significant and the contribution to total variation was zero. The Nm value was 0.85 (haploid) which shows very limited genetic exchange among the population. There was a very low number of individuals identified with admixture (FKB6, FKB7, and FAbUP-6) when analyzed with the admixture model using STRU CTU RE software (Fig. 4). All the individuals were stratified into two populations K = 2 as evidenced from the Evanno et al. (2005) method. Similar results were obtained from clustering using UPGMA. All the isolates from TN, A.P., Assam, and Gujarat fall under cluster I while others from U.P. and Bihar except FKB6, FKB7, and FAbUP7 were at the juncture which admixture model showed was admixed.

Discussion
Fusarium oxysporum f.sp. cubense TR4 has been reported from South America (Peru and Columbia), Africa (Mozambique), Asia, (Oman, Turkey, Israel, Jordan, India, Pakistan, China, Vietnam, Guangxi, Myanmar, Thailand, Guangdong, Fujian, Philippines, Taiwan, Malaysia, Sumatra, Java, Indonesia, Sulawesi) and Australia https:// www. cabi. org/ isc/ datas heet/ 59074 053# toDis tribu tionM aps). In India, it was first reported from Ayodhya, U.P. and Katihar, Bihar (Damodaran et al. 2018;Thangavelu et al. 2019); but now has been spread to other districts of the states where banana is popularly grown. All the symptoms noticed during the surveys were characteristic symptoms of wilt described by other workers across the world (Ploetz 2015;Pegg et al. 2019). Significant morphological variability in terms of size, septation, and abundance of micro and macro-conidia may arise due to the slight fluctuation in light, pH, temperature, carbon/nitrogen source, and exposure to UV radiation (Carlile 1956). In the case of Fusarium spp., PDA is preferred for studying the colony characteristics, however, the conidia size and shape don't remain consistent on the PDA substrate. Therefore, carnation leaf agar (CLA) and SNA media are preferred for examining spore characteristics (Leslie and Summerell 2006). Thus, in the current study, we have characterized the colony characters on PDA and spore characteristics on both PDA and SNA. On PDA, the mycelium of two isolates viz., FKB-6 and FKB-13 transformed phenotypically into flat wet white mycelium after 1-2 sub-culturing on PDA plates. Similar observations were made by Aguilar-Hawod et al. (2020). Such phenotypic variations have been attributed due to the mutations induced by transposable elements that affect the chromosome structures, gene sequences, and ultimately the gene expressions (Roncero et al. 2003;Groenewald et al. 2006;Aguilar-Hawod et al. 2020). The spore size and morphology of all the isolates were similar on both media (PDA and SNA), however the size of all the spores observed on SNA was slightly more as compared to the PDA media. The microconidia size and morphology remained consistent on both PDA and SNA but it was not the case with macroconidia on PDA. The morphological features of all 41 isolates were studied under uniform conditions; therefore the variations obtained in the case of these isolates are attributed to the genotype of a pathogen. All the isolates included in the pathogenicity assay were pathogenic on banana cv. Grand Naine (G9). The results were consistent with the studies carried out by Aguilar-Hawod et al. (2020). However, in the study conducted by Maymon et al. (2020), the TR4 isolates from Israel produced symptoms only after 14 days of inoculation and completely killed the plants after 4 weeks of inoculation.
Traditionally, the Foc races were identified through vegetative compatibility assays (VCG assays), which is a laborious, time taking process and its success depends on the availability of VCG tester lines, generation of nitM, and nit1/3 mutants. Currently, the Foc race specific molecular detection methods are available on public platform. Thangavelu et al. (2022) have designed Foc race specific primers for Indian Foc population targeting the SIX1a protein for TR4, SIX7a protein for STR4 and a hypothetical protein (XM_018394505.1) for Race 1. Using these primer pairs the isolates from TN, AP, Gujarat, and Assam were identified as race 1. Notably, these isolates were pathogenic on banana cv. G9 in pathogenicity assays, thus indicating the presence of virulent strain of Race 1 in these locations (Thangavelu and Mustaffa 2010;Thangavelu et al. 2021). The results were in parallel with the studies concluded by Thangavelu et al. (2012), Damodaran et al. (2018),    (Achari et al. 2023). Cluster analysis based on ISSR markers grouped the current isolates majorly in 2 clusters, all the TR4 isolates clustered in group A and Race1 isolates in group B. Thus, we could detect strong correlation between race structure and ISSR grouping in the present study. In accordance with the present study, Thangavelu et al. (2012) also obtained dendrogram based on ISSR markers in which Race4 isolates were clustered separately from Race1 isolates. Groenewald et al. (2006) assessed the genetic variability of Foc isolates collected from South Africa, Indonesia, Brazil, Malaysia, Australia, Phillipines, and Taiwan with AFLP analysis. In the dendogram inferred by AFLP, no correlation was observed between geographical location of the isolates, race structure, and AFLP clustering (Groenewald et al. 2006). Aguilar-Hawod et al. (2020) assessed the molecular variability of 42 isolates from Germany through Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR, Repetitive Extragenic Palindromic (REP)-PCR, and RAPD-PCR. Out of all PCR based markers, ERIC primers were the most informative as the correlation between race structure and grouping was obtained in the dendogram (Aguilar-Hawod et al. 2020). In the present study, the results obtained from UPGMA clustering through NTSYS were in good accordance with that of STRU CTU RE analysis. It is quite evident that significant genetic variation was observed in isolates from different states and isolates within the districts of states. Thus, genetic heterogeneity among TR4 isolates may compromise the effectiveness and precision of current TR4-specific diagnostic procedures in future. Therefore, it is crucial to regularly assess the efficacy of present diagnostics and develop powerful diagnostics if it is demonstrated that they are incapable of identifying TR4. This current study indicates the widespread occurrence of Foc TR4 in U.P. and Bihar states suggesting the need for stringent quarantine and management strategies to contain the spread of the pathogen to other banana growing regions of the country. Thus, it is recommended to contain the further spread of Foc TR4 to other major banana growing areas of U.P. as well as other states through avoiding the movement of banana planting materials across the fields, planting the healthy tissue culture raised banana plants and avoiding the plantation of rattoon crop. Additionally, it is also recommended for regular and frequent screening of banana plants from newer areas to ascertain the actual status of Foc TR4 spread and distribution in the country.
Author's contributions NK: methodology and investigation and writing-original draft preparation. IA: methodology and investigation. AK: AMOVA and Structure software. TD, SR: conceptualization, funding acquisition, Surveys, Map preparation. MM, PKS: Isolates collection, resources, review and editing. RG, KY, PB: Isolates collection. All authors contributed to the article and approved the submitted version.
Funding This work has been supported from Application of Microorganisms in Agriculture and Allied Sectors (AMAAS) project operated at ICAR-Central Institute of Soil Salinity Research, Regional Research Station, Lucknow and Flagship project of Indian Council of Agricultural Research operated at ICAR Central Institute for Subtropical Horticulture, Lucknow.

Declarations
Conflict of interest All authors declare that they have no conflict of interest.
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