Molecular identification, race differentiation and host differential analysis of Fusarium oxysporum f. sp. ciceri associated with chickpea wilt

Introduction

Chickpea (Cicer arietinum L.) is one of the most important pulse crops cultivated worldwide and serves as a major source of dietary protein, carbohydrates, vitamins and minerals particularly in developing countries. India is the largest producer of chickpea; however, its productivity is severely constrained by several biotic stresses among which Fusarium wilt, caused by Fusarium oxysporum f. sp. ciceri (FOC), is one of the most destructive diseases. The pathogen is both seed and soil borne and can survive in soil for several years through chlamydospores this making disease management difficult. Under favorable environmental conditions Fusarium wilt can cause yield losses ranging from 10 to 100% depending on the susceptibility of the cultivar and the virulence of the pathogen. The pathogen exhibits considerable pathogenic and genetic variability resulting in the occurrence of multiple physiological races that differ in their virulence and geographical distribution. The emergence of new races often overcomes host resistance, reducing the effectiveness of resistant cultivars and posing a major challenge to chickpea breeding programs. Therefore, understanding the diversity, pathogenicity and race structure of Fusarium oxysporum f. sp. ciceri populations is essential for developing durable disease management strategies.

Methods

Sample collection

Extensive field visits were conducted during the Rabi seasons of 2018-19 across diverse geographical regions and major chickpea-producing hubs in India (Table 1). Samples were collected from farmers' fields based on characteristic physiological symptoms, including:

• Marked wilting of the plant.

• Paler or greyish green foliage.

• Internal vascular necrosis (brown to black discoloration of the pith and xylem) observed upon root inspection in both seedling and adult stages. All symptomatic plant materials were carefully labelled and transported to the laboratory for further processing and pathogen isolation.

Isolation of the causal pathogen

Pathogen isolation was performed on PDA medium within a strictly aseptic environment. Initially the working surface of the laminar airflow cabinet was disinfected with denatured spirit. All necessary materials including petri plates, blades, needles, and forceps were placed inside and the Ultraviolet (UV) lamp was activated for 30 minutes prior to use. The primary or secondary roots and the collar region of the symptomatic chickpea plants were used for isolation. The procedure involved:

• Selected plant tissues were cut into small segments (10 to 20 mm) using a sterile razor. • Segments were submerged in a 0.1% mercuric chloride (HgCl₂) solution for one minute. • Bits were subjected to three subsequent rinses in sterile distilled water to eliminate any residual mercuric chloride.

• After being air dried on sterile tissue paper for two minutes five segments were placed equidistantly on pre poured PDA plates.

• Plates were incubated in a BOD incubator at 27 ± 2 °C for three to four days. • Upon the emergence of fungal growth the mycelia were aseptically transferred to PDA slants incubated for an additional 7 days at 27 ± 2 °C and subsequently stored in a refrigerator for further study.

Identification of the isolated pathogen

The identity of the Fusarium oxysporum f. sp. ciceri isolates was confirmed through:

• Morphological characterization: Observations were cross referenced with established descriptions by Booth (1975) [5] , Nelson et al. (1983) [23] , and Leslie & Summerell (2006) [21] . • Molecular validation: Genetic identification was conducted (Table 2) using Internal Transcribed Spacer (ITS) analysis with forward (ITS 1) and reverse (ITS 4) primers (White et al., 1990) [36] .

Race differentiation of isolates of Fusarium oxysporum f. sp. ciceri

The isolates of Fusarium oxysporum f. sp. ciceri were distinguished into the four races. Using the procedure outlined by Jimenez Gasco and Jimenez Diaz (2003) [19] , high quality and quantity DNA was isolated from the mycelium of Fusarium oxysporum f. sp. ciceri. In order to fingerprint the races of Fusarium oxysporum f. sp. ciceri, four primer pairs with ITS-specific markers were utilized (Table 3). The pathogenicity of the isolates was evaluated on the susceptible chickpea cultivar JG-62 using the soil inoculation method (Nene et al., 1981) [24] . The pathogen was multiplied on sand-maize meal medium (3:1, w/w), and inoculum was mixed into sterilized soil at 10 g kg⁻¹ soil seven days before sowing. Surface-sterilized seeds were sown in inoculated pots, while uninoculated pots served as controls. Wilt incidence was recorded 40 days after inoculation, and isolates were classified into pathogenicity groups according to Haware and Nene (1982a) [17] .

Disease assessment and data analysis

Inoculated seedlings were inspected every second day for disease symptoms. Wilt incidence was calculated based on the following formula: Wilt Incidence (%) = (Number of plants wilted / Total number of plants sown) × 100 Isolates were categorized based on the virulence scale proposed by Haware and Nene (1982a) [17] :

Non-pathogenic isolates (NPI) 0% ii)

Weakly pathogenic isolates (WPI) 1-20% iii)

Moderately pathogenic isolates (MPI) 21-50% iv)

Strong pathogenic isolates (SPI) 51-70% v)

Highly pathogenic isolates (HPI) >70%

2.8 Host differential analysis of Fusarium oxysporum f. sp. ciceri Forty representative isolates were evaluated on ten chickpea differential cultivars (JG-62, JG-74, C-104, CPS-1, BG-212, WR-315, Annigeri, Chaffa, L-550, and K-850) using the soil inoculation method. Disease incidence was recorded 40 days after inoculation, and host reactions were classified as resistant (0-20%), moderately susceptible (21-50%), or susceptible (>50%) based on percent wilt incidence.

Soil inoculation method

Sand maize meal medium (3:1, w/w) was used for mass multiplication of the pathogen inoculum. The medium was prepared by mixing 150 g of dry sand and 50 g of maize meal with 20 ml of distilled water in 1000 ml conical flasks.

The flasks were autoclaved at 1.05 kg cm⁻² pressure for 30 minutes for three consecutive days. After sterilization the sand maize meal medium was inoculated separately with pure cultures of Fusarium oxysporum f. sp. ciceri under aseptic conditions and incubated at room temperature for two weeks. Plastic pots (15 cm diameter) were surface sterilized with 0.1% mercuric chloride (HgCl 2 ) and filled with 2 kg of sterilized soil treated with 1.0% formalin for 15 days. The soil was inoculated with 15 days old fungal cultures grown on sand maize meal medium at the rate of 10 g kg⁻¹ soil seven days prior to sowing. Seeds of each test cultivar were surface disinfected by immersion in a 2.5% sodium hypochlorite solution for 2-3 minutes before sowing. Ten seeds of each cultivar were sown per pot. Uninoculated pots were maintained as controls for each cultivar. The plants were observed periodically after sowing and seedlings were monitored for the initiation of wilt symptoms. Disease observations were recorded 40 days after inoculation.

Results and Discussion

Collection of disease samples

Chickpea wilt sample collection was carried out during Rabi 2018 and 2019 season on 118 different locations in major chickpea growing region of the country belonging to diverse Agro Ecological Regions (Table 4).

Isolation and purification 3.2.1 Isolation

During the survey diseased chickpea plants exhibiting typical wilt symptoms were collected from different Agro Ecological Regions and brought to the laboratory for pathogen isolation. The tissue isolation method was employed for the recovery of Fusarium oxysporum f. sp. ciceri from infected stem and root portions.

Infected tissues were cut into small segments and surface sterilized with 0.1% mercuric chloride solution for 40-60 seconds followed by two rinses in sterile distilled water to remove traces of the sterilant. The sterilized tissue segments were transferred onto potato dextrose agar (PDA) medium under aseptic conditions and incubated at 27 ± 2 °C in a BOD incubator to allow fungal growth.

Based on cultural and morphological characteristics, isolates of Fusarium oxysporum f. sp. ciceri were purified and maintained on PDA slants at 4 °C for further studies. A total of 118 isolates were successfully obtained and designated sequentially as S-1 to S-118, as presented. [39] . The frequent occurrence of Trichoderma spp. Also suggest natural antagonism in certain samples. Potentially explaining reduced FOC recovery in some locations as reported by Sharma et al., (2016) [32] . Similarly, competitive saprophytes like Aspergillus spp. and Rhizoctonia spp. may suppress or mask FOC during isolation under in vitro conditions.

Rationale for selecting 82 isolates for pathogenicity testing

Only those 82 samples that yielded confirmed Fusarium oxysporum f. sp. ciceri were selected for pathogenicity testing. Samples that showed 0% yield were excluded because pathogenicity assays require viable and pure cultures of the pathogen to fulfil Koch's postulates (Agrios, 2005) [1] . Without successful isolation and purification disease causation cannot be experimentally validated. Selection based on successful recovery is a standard procedure in wilt pathogen studies (Haware and Nene, 1982; Jiménez-Díaz et al., 2015) [17,39] where only confirmed isolates are advanced for virulence assessment. This ensures:

1) Experimental reliability and reproducibility 2) Elimination of false negatives due to absence of pathogen 3) Accurate assessment of virulence diversity 4) Avoidance of interference from non-target fungi Thus, the 82 isolates selected for pathogenicity testing represent the active FOC population recovered from diverse geographic regions. The variation in isolation frequency further suggests heterogeneity in pathogen distribution and inoculum density across chickpea growing areas. Overall, the isolation data confirm widespread distribution of Fusarium oxysporum f. sp. ciceri also highlighting variability in pathogen prevalence and soil mycoflora composition.

Purification of Fusarium oxysporum f. sp. ciceri isolates by Single spore isolation method

The isolates of Fusarium oxysporum f. sp. ciceri were purified using the single spore isolation technique to obtain genetically uniform cultures. Spore suspensions were prepared from actively growing cultures and spread onto 2% water agar medium. Spore germination was observed under a microscope within approximately 4 hours of inoculation. Individual germinated spores (8-12 hours after inoculation) were carefully selected and transferred aseptically onto fresh Potato Dextrose Agar (PDA) medium. Visible colonies developed within 2-3 days depending on the isolate. The use of single spore isolation ensured genetic purity and uniform colony growth compared to cultures obtained through mass transfer of inoculum (Leslie and Summerell, 2006) [21] . A thin layer of water agar facilitated clear microscopic observation and easy transfer of germinated spores using a sterile needle under low magnification. During standardization it was observed that spore densities exceedingly approximately 10 spores per microscopic field resulted in overlapping germ tubes making isolation difficult. Therefore, serial dilutions (10⁻³ and higher) were prepared to obtain well separated germinating spores for accurate isolation. The pathogenic variability of Fusarium oxysporum f. sp. ciceri isolates was assessed using the susceptible chickpea cultivar JG-62 under controlled conditions. A total of 82 isolates were evaluated with ten plants inoculated per isolate and wilt incidence was recorded to determine percent disease expression and categorize virulence. Wilt incidence ranged from 0 to 100 percent indicating considerable pathogenic diversity within the population. Highly Pathogenic Isolates (HPI; > 71% wilt) constituted a major proportion of the population with several isolates (S-8, S-69, S-70, S-78 and S-111) causing 100% wilt and complete plant mortality. Strongly Pathogenic Isolates (SPI; 51-70%) also represented a significant group while moderate (MPI; 21-50%) and weakly pathogenic isolates (WPI; 1-20%) showed reduced virulence. Two isolates (S-37 and S-41) were non-pathogenic (0% wilt). Overall, the predominance of HPI and SPI categories suggests the presence of highly aggressive strains within the tested population.

Pathogenic viability

At the end of the experiment Fusarium oxysporum f. sp. ciceri was successfully re-isolated from wilted plants, and its morphological characteristics resembled those of the original cultures thereby confirming Koch's postulates.

The virulence and pathogenic variability of Fusarium oxysporum f. sp. ciceri on cv. JG -62 have been widely documented (Prasad and Padwick, 1939 [27,16,20,34,3,33,8] . Similar differentiation among large numbers of isolates has been reported by Gupta et al.

(1986b) [15] who characterized 86 isolates collected from 84 locations of Madhya Pradesh. Paulkar et al. (2002) [25] and Saabale and Dubey (2014) [30] also reported substantial variability with wilt incidence ranging from 24 to 100 percent among isolates tested on JG -62 under pot conditions. Golakiya et al. (2018) [14] further confirmed pathogenic variability among fifteen isolates screened on the susceptible cultivar. Grouping of isolates based on pathogenicity revealed no consistent relationship between geographical origin and virulence indicating that pathogenic potential is not strictly region dependent. Similar observations were reported by Durai et al. (2012) [9] who found that isolates from different regions were pathogenic irrespective of their origin. The classification of Fusarium oxysporum f. sp. ciceri isolates based on pathogenic ability has been documented earlier by Prasad and Padwick (1939) [27] , El-Morsey et al. (1997) [10] , Rao and Krishnappa (1997) [30] , Zamani et al. (2000) [38] , Shubha Trivedi and Gurha (2005) [34] and Barhate et al.

(2006) [3] . The wide spectrum of virulence observed in the present study confirms significant pathogenic diversity within Fusarium oxysporum f. sp. ciceri populations. The predominance of highly virulent isolates highlights the potential threat to chickpea cultivation and underscores the importance of continuous monitoring of virulence patterns and deployment of durable resistance strategies. [17,24] . Prioritizing highly virulent isolates ensures accurate assessment of host resistance and reflects the worst case disease scenario under field conditions (Agrios, 2005) [1] . [39,32] . Thus, the stepwise selection (118 → 82 → 40) ensured biological validity, virulence relevance and broad geographic representation, making these isolates suitable for further evaluation studies.

Identification of Fusarium oxysporum f. sp. ciceri isolates by ITS marker

In the present study universal primers ITS-1 (5′ TCC GTA GGT GAA CCT GCG G 3′) and ITS-4 (5′ TCC TCC GCT TAT TGA TAT GC 3′) were employed to amplify the ITS1-5.8S-ITS2 region. PCR amplification was carried out in a 50 µL reaction volume following the protocol described by Gautam et al. (2013) [11] . The amplification of a single ~ 550 bp fragment confirmed the identity of all 40 isolates as Fusarium oxysporum f. sp. ciceri. These selected (40) isolates were used for further studies.

Identification of Fusarium at the species level can often be achieved based on cultural and morphological characteristics (Booth, 1971; Gerlach and Nirenberg, 1982; Nelson et al., 1983) [4,13,23] . Accordingly, all isolates were subcultured on Potato Dextrose Agar (PDA) and examined microscopically to confirm their identity and ensure purity prior to further analysis (Prasad et al., 2008) [28] . Subsequently the 40 highly pathogenic isolates of Fusarium oxysporum f. sp. ciceri were confirmed at the molecular level using ITS-1 and ITS-4 universal primers (White et al., 1990) [36] . The ITS primer pair consistently amplified a single DNA fragment of approximately 550 bp in all tested isolates confirming their identity. Similar amplification patterns (~550 bp) for Fusarium oxysporum f. sp. ciceri using ITS primers have been reported by Gayatri Gurjar et al. (2009) [12] . The utility of ITS markers for molecular identification and characterization of Fusarium species has also been validated in earlier studies (Dubey et al., 2010; Durai et al., 2012; Datta and Lal, 2012) [7,9,6] . Based on molecular confirmation and virulence profiling representative isolates were further selected for molecular variability analysis to assess genetic diversity within the pathogen population. Manikandan and Raguchander (2014) [22] utilized internal transcribed spacer (ITS) regions for rapid identification and characterization of twenty isolates of Fusarium oxysporum f. sp. lycopersici. Amplification using ITS-1 and ITS-4 primers consistently produced a fragment of approximately 560 bp in all isolates, demonstrating the reliability of ITS markers for detection of Fusarium wilt pathogens. Similarly, Wu Kai li et al. (2019) [37] investigated Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense (FOC) and identified isolates using ITS sequence analysis. Among 33 putative Fusarium isolates ITS sequencing differentiated species effectively nineteen isolates were identified as Fusarium oxysporum eight as Fusarium solani, three as Fusarium proliferatum one as Fusarium verticillioides, while two remained unidentified. Their findings further support the utility of ITS sequencing in species level discrimination within the Fusarium complex.

Collectively these studies validate the use of ITS-based molecular markers as a reliable, rapid, and widely accepted approach for accurate identification of Fusarium spp. and formae speciales.

Racial identification of Fusarium oxysporum f. sp. ciceri isolates by molecular FDP marker

Molecular characterization of Fusarium oxysporum f. sp. ciceri (FOC) races has been widely undertaken using race specific DNA markers targeting ITS regions and other gene sequences. Poornima et al. (2017) [26] differentiated four major races of Fusarium oxysporum f. sp. ciceri prevalent in India using race specific primers FDP-3, FDP-14, FDP-2, and FDP-9.

Primer FDP-3 targets the mitochondrial rRNA gene and produces a polymorphic banding pattern around 700 to 720 bp, which is characteristic of race 1. This race has been predominantly reported from Maharashtra, Karnataka and Andhra Pradesh. Here, in our study isolates FOC-1 to FOC- [26] , FDP-2 (ITS region), FDP-3 (mitochondrial rRNA gene), FDP-9 (race 4specific), and FDP-14 (race 2-specific) were found to be reliable for distinguishing Indian races. Earlier demonstrated the effectiveness of mitochondrial rRNA-based primers in fungal differentiation. Furthermore, EST based markers and translation elongation factor (EF-1α) genes have also been used for assessing variability among Fusarium isolates (Arif et al., 2012) [2] . Globally, eight races of Fusarium oxysporum f.sp ciceri (0, 1A, 1B/C, 2, 3, 4, 5, and 6) have been reported of which four races (1, 2, 3, and 4) are predominant in India (Venkataramanamma et al., 2020) [35] . Race 1 has been particularly reported from Andhra Pradesh. The emergence of new races poses a serious threat to chickpea cultivation, as it may overcome existing host resistance. Therefore, continuous monitoring of newly collected isolates from diverse geographical regions and host genotypes is essential to understand racial distribution and evolution. Overall, race specific molecular markers provide a rapid, reliable and precise method for differentiating Fusarium oxysporum f. sp ciceri races and are crucial for resistance breeding and disease management strategies.

Assaying the isolates reaction on the basis of host differential studies in vitro

7.1 Host differential analysis of Fusarium oxysporum f. sp. ciceri An experiment was conducted to evaluate host differential reactions of forty isolates of Fusarium oxysporum f. sp. ciceri collected from different chickpea growing regions of India. The isolates were screened on ten international chickpea differential cultivars, namely JG-62, C-104, JG-74, CPS-1, BG-212, WR-315, Annigeri, Chaffa, L-550, and K-850, using the soil inoculation method. Sterilized soil was artificially inoculated with sand-maize meal inoculum of Fusarium oxysporum f. sp. ciceri at 3:1 percent (w/w). Ten seeds of each differential cultivar were sown in pots inoculated with individual isolates. After germination, five healthy seedlings per pot were maintained. Observations on days to initial wilting and complete wilting were recorded up to 40 Days After Sowing (DAS). Seedlings grown in sterilized uninoculated soil served as control.

Differential reactions

The susceptible check JG-62 exhibited susceptible (S) reaction to all forty isolates belonging to Race [8] characterized seventy isolates from thirteen states into eight races using differential cultivar responses and identified specific cultivar combinations distinguishing races 1 through 8. Similarly, Ramanamma et al. (2020) [29] reported the occurrence of four races (1, 2, 3, and 4) in India and confirmed the prevalence of race 1 in Andhra Pradesh. The present investigation confirms the existence of multiple races (race 1, 2, 3, and 4). Among the tested isolates and demonstrates substantial pathogenic and racial variability across chickpea growing regions. The differential reactions observed in this study provide important insights for resistance breeding programs and support the development of region-specific wilt-resistant chickpea varieties.

Conclusion

1) A total of 118 chickpea wilt samples were collected from different agro ecological regions of India out of which 82 isolates of Fusarium oxysporum f. sp. ciceri (FOC) were successfully isolated and purified. 2) Pathogenicity testing revealed considerable variability in virulence with wilt incidence ranging from 0 to 100%, indicating the existence of diverse pathogen populations. 3) Based on pathogenicity, 40 highly virulent isolates (80-100% wilt incidence) were selected for detailed molecular characterization. 4) Molecular identification using ITS1 and ITS4 primers confirmed all selected isolates as Fusarium oxysporum f. sp. ciceri by producing a characteristic ~550 bp amplicon. 5) Race specific molecular markers successfully differentiated the isolates into four races (Race 1, Race 2, Race 3, and Race 4), with Race 1 being the predominant race. 6) Host differential analysis demonstrated clear differences in cultivar reactions, confirming the presence of distinct pathogen races and pathogenic variability. 7) The chickpea cultivar WR-315 exhibited broadspectrum resistance against all tested races, whereas JG-62 was susceptible to all isolates. 8) No consistent relationship was observed between the geographical origin of the isolates and their pathogenicity, indicating that virulence is independent of collection location. 9) The combined use of pathogenicity assays, ITS based molecular identification, race specific markers and host differential analysis proved effective for comprehensive characterization of FOC isolates. 10) The findings provide valuable information for race monitoring, identification of resistant germplasm, resistance breeding and the development of sustainable management strategies against Fusarium wilt of chickpea.

References

1. Agrios GN. Plant pathology. 5 th ed. Burlington (MA): Elsevier Academic Press; 2005.
2. Arif M, Chawla S, Zaidi NW, Rayar JK, Variar M, Singh US. Development of EST markers for genetic diversity analysis in Fusarium oxysporum. Mol Biotechnol. 2012;52:93-100.
3. Barhate BG, Patil PV, Raut BT. Pathogenic variability in Fusarium oxysporum f. sp. ciceri isolates from Maharashtra. J Plant Dis Sci. 2006;1:115-118.
4. Booth C. The genus Fusarium. Kew, Surrey: Commonwealth Mycological Institute; 1971.
5. Booth C. The genus Fusarium. Kew, Surrey: Commonwealth Mycological Institute; 1975. 237 p.
6. Datta S, Lal HC. Molecular characterization of Fusarium oxysporum f. sp. ciceri using ITS markers. Indian Phytopathol. 2012;65:318-322.
7. Dubey SC, Singh SR, Singh B. Morphological and molecular characterization of Fusarium oxysporum f. sp. ciceri isolates. Indian Phytopathol. 2010;63:451457.
8. Dubey SC, Singh SR, Singh B. Morphological and pathogenic variability of Fusarium oxysporum f. sp. ciceri isolates causing chickpea wilt in India. Arch Phytopathol Plant Prot. 2012;45:174-190.
9. Durai R, Nakkeeran S, Renukadevi P. Morphological and pathogenic variability among isolates of Fusarium oxysporum f. sp. ciceri collected from southern India. Arch Phytopathol Plant Prot. 2012;45:977-986.
10. El Morsey SA, Abdel Monaim MF. Pathogenic variability among isolates of Fusarium oxysporum f. sp. ciceris. J Phytopathol. 1997;145:297-302.
11. Gautam AK, Avasthi S, Bhadauria R. Molecular identification of Fusarium species using ITS-rDNA sequence analysis. Afr J Biotechnol. 2013;12:502-510.
12. Gurjar G, Gopalakrishnan C, Punja ZK. Identification of Fusarium oxysporum f. sp. ciceri using ITS-PCR markers. J Plant Pathol. 2009;91:151-158.
13. Gerlach W, Nirenberg H. The genus Fusarium: a pictorial atlas. Berlin-Dahlem: Mitteilungen aus der Biologischen Bundesanstalt für Landund Forstwirtschaft; 1982.
14. Golakiya BA, Khandar RR, Patel DS. Pathogenic variability among isolates of Fusarium oxysporum f. sp. ciceri infecting chickpea. J Pharmacogn Phytochem. 2018;7(5):2674-2678.
15. Gupta OM, Kotasthane SR, Khare MN. Pathogenic variability in Fusarium oxysporum f. sp. ciceri isolates from Madhya Pradesh. Indian Phytopathol. 1986;39:581-584.
16. Haware MP, Nene YL. Races of Fusarium oxysporum f. sp. ciceri. Plant Dis Rep. 1979;63:343-346.
17. Haware MP, Nene YL. Races of Fusarium oxysporum f. sp. ciceri. Plant Dis. 1982;66:809-810.
18. Jimenez-Diaz RM, Castillo P, Jimenez-Gasco MM, Landa BB, Navas-Cortes JA. Fusarium wilt of chickpeas: biology, ecology and management. Crop Prot. 2015;73:16-27.
19. Jimenez-Gasco MM, Jimenez-Diaz RM. Development of race-specific polymerase chain reaction markers for race 0, race 1B/C, race 5, and race 6 of Fusarium oxysporum f. sp. ciceris and their use in genetic diversity studies. Phytopathology. 2003;93(2):200-209. International Journal of Agriculture and Food Science https://www.agriculturaljournals.com
20. Kewate SD. Studies on Fusarium wilt of chickpea. Akola (India): Dr. Panjabrao Deshmukh Krishi Vidyapeeth; 1986.
21. Leslie JF, Summerell BA. The Fusarium laboratory manual. Ames (IA): Blackwell Publishing; 2006. 388 p. 22. Manikandan R, Raguchander T. Molecular characterization of Fusarium oxysporum f. sp. lycopersici isolates using ITS markers. Arch Phytopathol Plant Prot. 2014;47:1904-1915.
22. Nelson PE, Toussoun TA, Marasas WFO. Fusarium species: an illustrated manual for identification. University Park (PA): Pennsylvania State University Press; 1983. 193 p.
23. Nene YL, Haware MP, Reddy MV. Chickpea diseases: resistance screening techniques. Information Bulletin No. 10. Patancheru (India): International Crops Research Institute for the Semi-Arid Tropics; 1981.
24. Paulkar PK, Khare MN, Kotasthane SR. Pathogenic variability in Fusarium oxysporum f. sp. ciceri isolates collected from central India. J Mycol Plant Pathol. 2002;32:128-131.
25. Poornima R, Rangeshwaran R, Karthikeyan G, Pramesh D. Development and validation of race-specific molecular markers for identification of Indian races of Fusarium oxysporum f. sp. ciceri. J Appl Microbiol. 2017;122:1200-1212.
26. Prasad N, Padwick GW. The genus Fusarium as a cause of wilt disease of gram (Cicer arietinum L.) in India. Indian J Agric Sci. 1939;9:371-380.
27. Prasad P, Gupta OM, Khare MN. Morphological and cultural characterization of Fusarium oxysporum f. sp. ciceri. J Mycol Plant Pathol. 2008;38:149-153.
28. Ramanamma AV, Venkataramanamma K, Reddy NPE. Distribution and characterization of races of Fusarium oxysporum f. sp. ciceri in India. Indian Phytopathol. 2020.
29. Rao MSL, Krishnappa M. Pathogenic variability in Fusarium oxysporum f. sp. ciceri isolates from Karnataka. Karnataka J Agric Sci. 1997;10:548-552.
30. Saabale PR, Dubey SC. Pathogenic and cultural variability among isolates of Fusarium oxysporum f. sp. ciceri. Indian Phytopathol. 2014;67:183-188.
31. Sharma M, Ghosh R, Pande S. Current status of wilt/root rot diseases of chickpea and their management. Arch Phytopathol Plant Prot. 2016;49(910):222-238.
32. Shrivastava AK, Agrawal SC. Variability among isolates of Fusarium oxysporum f. sp. ciceri causing chickpea wilt. J Mycol Plant Pathol. 2006;36:220-223.
33. Trivedi S, Gurha SN. Pathogenic variability among isolates of Fusarium oxysporum f. sp. ciceri causing chickpea wilt. J Mycol Plant Pathol. 2005;35:238-241.
34. Venkataramanamma K, Poornima R, Rangeshwaran R, Pramesh D. Distribution and molecular characterization of races of Fusarium oxysporum f. sp. ciceri in India. Indian Phytopathol. 2020;73:473-482.
35. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols: a guide to methods and applications. San Diego (CA): Academic Press; 1990. p.315-322.
36. Wu K, Li Y, Zhang X, Wang Y, Chen J. Identification and characterization of Fusarium oxysporum isolates associated with banana wilt using ITS sequence analysis. Plant Dis. 2019;103:2063-2072.
37. Zamani MR, Motallebi M. Pathogenic and genetic variability among isolates of Fusarium oxysporum f. sp. ciceri. J Phytopathol. 2000;148:419-425.
38. Jiménez-Díaz RM, Castillo P, Jiménez-Gasco MM, Landa BB, Navas-Cortés JA. Fusarium wilt of chickpeas: biology, ecology and management. Crop Prot. 2015;73:16-27. DOI:10.1016/j.cropro.2015.02.023.