Evaluation of commercial varieties and hybrids for resistance to gummy stem blight in watermelon

Introduction

Watermelon (Citrullus lanatus [Thunb.] Matsum. & Nakai) is an economically important cucurbit cultivated extensively across tropical and subtropical regions for its nutritional and commercial value (Wehner, 2008) [18] . India is one of the major producers of watermelon, and the crop contributes significantly to farm income, particularly in arid and semi-arid regions. However, watermelon production is constrained by several fungal, bacterial, and viral diseases, among which gummy stem blight (GSB) is considered one of the most destructive (Keinath, 2014) [7] . Gummy stem blight is caused by a species complex within the genus Stagonosporopsis (formerly Didymella bryoniae), primarily S. cucurbitacearum, S. citrulli, and S. caricae (Stewart et al., 2015) [13] . These pathogens infect stems, leaves, petioles, and fruits, producing characteristic water-soaked lesions, necrosis, stem cankers, and gummy exudates (Keinath & DuBose, 2004) [8] . Severe infections can lead to rapid vine collapse, premature drying, and economic losses in both field and greenhouse-grown crops. Environmental conditions play a critical role in disease development. High relative humidity (>90%), extended leaf wetness, and moderate temperatures (20-28 °C) favor infection, sporulation, and disease spread (Zitter, Hopkins & Thomas, 1996) [7] . The pathogen is seedborne and survives in crop debris, allowing it to persist between seasons and initiate new infections (Schenck, 1968) [11] . Due to its polycyclic nature and multiple infection cycles within a growing season, GSB is difficult to manage using fungicides alone. Chemical control, although widely practiced, is increasingly challenged by fungicide resistance. Resistance to QoI (strobilurin) and MBC (benzimidazole) fungicides in Stagonosporopsis spp. has been reported from several watermelon-growing regions worldwide (Avenot et al., 2012; Keinath, 2011) [1,6] . In addition, reliance on repeated fungicide applications raises concerns related to cost, environmental impact, and residue accumulation. Consequently, host plant resistance is considered the most sustainable and effective management strategy for GSB (Boyhan et al., 2014) [2] . Despite the importance of resistant cultivars, information on the resistance status of commercially cultivated watermelon hybrids in India remains limited. Previous studies have highlighted inconsistencies in resistance assessment due to variability across inoculation methods such as leaf spray, root inoculation, and leaf disk placement (Chiu and Walker, 1949; Van Der Meer et al., 1978; Wyszogrodzka et al., 1986) [4,14,20] . Therefore, identifying a reliable inoculation method and systematically evaluating available hybrids are essential for accurate identification of resistance sources. The present study was undertaken to compare different artificial inoculation methods for their reliability in producing uniform symptoms of gummy stem blight, and screen twenty commercial varieties and hybrids of watermelon for resistance to Stagonosporopsis cucurbitacearum under controlled polyhouse conditions.

The findings aim to identify promising genotypes for resistance breeding and integrated management of gummy stem blight in watermelon.

Materials and Methods

Plant material and experimental setup

Twenty commercial watermelon (Citrullus lanatus) varieties and hybrids were obtained from public and private seed companies and research institutes for resistance screening against gummy stem blight (Table 1). Seeds were sown in plastic pots containing sterilized soil, with five seeds per pot and one pot considered as a single replication; each variety/hybrid was replicated three times under greenhouse conditions (25-30 °C) without application of any fungicides or other chemical sprays. For seedling-stage pathogenicity and method comparison experiments, surface-sterilized seeds of each entry were sown in protrays (98 cells) filled with cocopeat and maintained in a greenhouse at ambient temperature until use.

Inoculation methods

Four inoculation methods were evaluated for screening: (i) root inoculation, (ii) leaf inoculation using mycelial discs, (iii) foliar spray of spore suspension, and (iv) stem injection of spore suspension.

Root inoculation

Ten-day-old seedlings raised in cocopeat were carefully uprooted, and roots were gently washed under running tap water to remove adhering substrate. Root systems were trimmed to approximately 2 cm length and dipped for 3 min in the fungal inoculum (mycelial or spore suspension), after which seedlings were transplanted into pots containing sterilized soil. Control seedlings were treated similarly but dipped in sterile distilled water. Plants were maintained in the greenhouse under ambient conditions.

Leaf inoculation with mycelial discs

For leaf inoculation, 15-day-old seedlings were used. Prior to inoculation, plants were irrigated to ensure full turgor.

One mycelial disc (cut from the actively growing margin of a 7-day-old SC-W10 culture) was placed on each of two fully expanded true leaves per seedling using a sterile toothpick, ensuring close contact with the leaf surface.

Cotyledons were not inoculated because cotyledon inoculation has been reported to be unreliable for assessing resistance to gummy stem blight. Inoculated seedlings were immediately covered with transparent polyethylene sheets to maintain high humidity (relative humidity >90% for the first 24 h and >70% for the following 7 days), using misting/fogging facilities as required. The average temperature in the polyhouse was maintained at 25-28 °C.

Foliar spray and stem injection inoculation

Pathogenicity tests and screening of entries were also performed on 15-day-old healthy seedlings using a spore suspension prepared from SC-W10 cultures. Seedlings were inoculated by:  Spraying the spore suspension onto the foliage with a hand sprayer until runoff.  Injecting the spore suspension into the stem using a sterile hypodermic syringe (stem injection method).

Control plants were sprayed and injected with sterile distilled water only. Following inoculation, plants were placed in a humidity chamber and maintained at relative humidity >90% until symptom development. The spore suspension was reapplied at two-day intervals until characteristic gummy stem blight symptoms appeared.

To confirm pathogenicity and fulfill Koch's postulates, the pathogen was re-isolated from symptomatic tissues of inoculated plants onto PDA and compared morphologically with the original SC-W10 culture.

Disease assessment and percent disease index

Disease severity on individual seedlings was recorded using the 0-9 categorical scale described by Gusmini et al. (2005) [5] :

Disease assessment and percent disease index The percent disease index (PDI) was calculated from disease ratings using McKinney's (1923) [9] formula:

The mean PDI for each variety/hybrid and inoculation method was used to categorize the reaction to gummy stem blight.

Results and Disussion

Disease development increased markedly with increasing relative humidity, and plants maintained at >90% RH exhibited greater pathogenicity and higher infection rates than those maintained at 70 ± 5% RH. Under these favorable conditions, all three inoculation methods tested foliar spraying of mycelial suspension, root inoculation, and placement of mycelial discs on true leaves were capable of causing infection, but they differed in reliability and symptom expression. Spraying a mycelial suspension onto the leaf surface produced erratic symptom expression and highly variable disease progression, whereas root inoculation frequently caused rapid plant collapse, limiting its usefulness for differential scoring. Inoculation by placing mycelial discs on true leaves at 95 ± 5% RH resulted in relatively slow, localized symptom development, while the stem injection method produced a uniform, progressive infection along the stem with clear, scorable lesions and no non-specific symptom development among the twenty genotypes tested. On this basis, disease assessment was most convenient and reproducible using the stem injection method, which was therefore adopted for subsequent screening. The details of disease incidence among the various hybrids/varieties during screenings under polyhouse conditions are presented in Table (2). After stem injection, all the hybrids/varieties started showing disease symptoms like a water-soaked lesion on leaves turning light brown in colour on the third day (Plate 2). Within seven days, lesions spread and started entering the leaf petiole in susceptible varieties. With the progress of time, the pathogen was able to penetrate the stem causing stem lesions. These lesions slowly dried up. The progress of infection was found to spread towards the bottom rather than the top of the plant. The disease score was recorded at seven-day intervals after inoculation. Observations were continued up to the 7 th day.

Based on the recorded disease score, percent Disease Index (PDI) was calculated. Among the 20 hybrids/varieties evaluated under polyhouse conditions, nine hybrids IB- [5] . GSB has been earlier reported to be exhibiting significant variability in terms of disease expression depending upon the conditions of screening in cucumber (Wehner and Shetty, 2000) [19] , melon (Zhang et al., 1997) and watermelon (Boyhan et al., 1994) [3] . This is because GSB infections are highly influenced by environmental conditions such as temperature, relative humidity and ventilation (Van Steekelenburg, 1984, 1985a; Van Steekelenburg and Vooren, 1980) [15,17,16] . The aggressiveness of a pathogen, its ability to infect disease scoring method along with environmental factors such as temperature and humidity can influence the resistance evaluation (Ren et al., 2020) [10] . Similarly, a decrease in the level of resistance of "PI 189225" was observed by Ren et al. (2020) [10] when kept under 100% RH after inoculation for a period of 48 h compared to 24 h. Therefore, to minimize the error and generate robust screening data, testing in multiple environments will be needed.

Plate 2: Disease incidence among the various hybrids/varieties during screenings under polyhouse conditions

Conclusion

These findings underline the ongoing need to identify and deploy cultivars with improved resistance for sustainable management of GSB, particularly due to the limitations and risks associated with chemical control. The work also highlights the crucial influence of environmental conditions, especially humidity, on disease expression and the accuracy of resistance evaluation. Ultimately, the results provide valuable insights for breeding programs, suggesting that screening under high-humidity, polyhouse conditions using stem injection can efficiently identify genotypes with moderate resistance, which can be vital components in integrated disease management strategies and resistance breeding for gummy stem blight in watermelon.

References

1. Avenot HF, Morgan DP, Michailides TJ. Resistance to QoI fungicides in Didymella bryoniae populations. Plant Disease. 2012;96(5):668-675.
2. Boyhan G, Granberry D, Kelley W. Watermelon production. University of Georgia Extension Bulletin. 2014;996:1.
3. Boyhan G, Norton JD, Abrahams BR. Screening for resistance to anthracnose (race 2), gummy stem blight, and root knot nematode in watermelon germplasm. Cucurbit Genetic Cooperative. 1994;17:106-110.
4. Chiu WF, Walker JC. The relationship of inoculation methods to disease development in gummy stem blight of cucurbits. Phytopathology. 1949;39:17-22.
5. Gusmini G, Song R, Wehner TC. New sources of resistance to gummy stem blight in watermelon. Crop Science. 2005;45(2):582-588.
6. Keinath AP. Sensitivity of Didymella bryoniae to fungicides. Crop Protection. 2011;30(9):1105-1111.
7. Keinath AP. Gummy stem blight. In: Zitter TA, Hopkins DL, Thomas CE, editors. Compendium of Cucurbit Diseases. APS Press; 2014. p. 45-47.
8. Keinath AP, DuBose VB. Evaluation of watermelon cultivars for gummy stem blight resistance. HortScience. 2004;39(6):1355-1358.
9. McKinney HH. Influence of soil temperature and moisture on infection of wheat seedlings by Helminthosporium sativum. USDA Publications. 1923;195-218.
10. Ren R, Xu J, Zhang M, Liu G, Yao X, Zhu L, et al. Identification and molecular mapping of a gummy stem blight resistance gene in wild watermelon (Citrullus amarus) germplasm PI 189225. Plant Disease. 2020;104(1):16-24.
11. Schenck NC. Survival of Mycosphaerella melonis. Phytopathology. 1968;58:123-124.
12. Sowell G, Pointer GR. Gummy stem blight resistance of introduced watermelons. Plant Disease Reporter. 1962;46:883-885.
13. Stewart JE, Turner AL, Brewer MT, Keinath AP. Species delimitation in the gummy stem blight pathogen species complex. Fungal Biology. 2015;119(6):442-456.
14. Van der Meer QP, de Waard MA, Wouters D. Screening for resistance to gummy stem blight in cucurbits. Netherlands Journal of Plant Pathology. 1978;84:141-152.
15. Van Steekelenburg NAM. Influence of ventilation temperature and low ventilation rates on incidence of Didymella bryoniae in glasshouse cucumbers. Acta Horticulturae. 1984;156:187-197.
16. Van Steekelenburg NAM, Van de Vooren J. Influence of the glasshouse climate on development of diseases in a cucumber crop with special reference to stem and fruit rot caused by Didymella bryoniae. WPGC. 1980;118:45-56.
17. Van Steekelenburg NAM. Influence of humidity on incidence of Didymella bryoniae on cucumber leaves and growing tips under controlled environmental conditions. Netherlands Journal of Plant Pathology. 1985;91:277-283.
18. Wehner TC. Watermelon. In: Prohens J, Nuez F, editors. Vegetables II: Handbook of Plant Breeding. Springer; 2008. p. 381-418.
19. Wehner TC, Shetty NV. Screening the cucumber germplasm collection for resistance to gummy stem blight in North Carolina field tests. HortScience. 2000;35:1132-1140.
20. Wyszogrodzka A, Verhoeff K, Wouters D. Evaluation of inoculation techniques for resistance screening. Plant Pathology. 1986;35:105-110.