Identification and fungicide sensitivity of pathogens causing Cucurbitaceae root rot in Zhejiang province

Abstract：【Objective】Cucurbitaceae is one type of the most important economic plants in the world, including watermelon, melon, gourd, cucumber, pumpkin and other common vegetables and fruits. With the increase of protected cultivation area and the increasingly serious land continuous cropping obstacles, soil-borne diseases of Cucurbitaceae have occurred seriously, affecting the yield and quality. Grafting technology is one of the effective ways to solve soil-borne diseases like Fusarium wilt. However, in recent years, watermelon and other melon crops have withered and died in many Cucurbitaceae production areas in Zhejiang, including watermelon grafted on gourd or pumpkin rootstocks. It is preliminarily determined that the plants may be infected with other soil-borne diseases other than fusarium wilt, but the specific type of pathogen has not been determined. Therefore, this study was conducted to investigate the soil-borne disease of Cucurbitaceae in Zhejiang.【Methods】The pathogens were isolated by tissue separation method, and small pieces of necrotic root tissues were cut, sterilized, dried and placed on PDA with streptomycin, growing at 25 ℃ for 4 d. The purified stains were stored on PDA at 4 ℃. The main pathogens causing Cucurbitaceae root rot were identified by morphological characteristic observa-tion, sequence analysis and pathogenicity test. The main causal agent of F. solani root rot from necrotic roots of watermelon, melon, bottle gourd and pumpkin was identified in Zhejiang. The pathogenicity of stains to different Cucurbitaceae crops was determined by yellowing seedling hypocotyl inoculation. The pathogens were inoculated on PDA containing penicillin and rifampicin for 5 d in dark at 25 ℃, and then the mycelium block was inoculated on the hypocotyl of the seedling with yellowing flattened cotyledons growing in dark, and wrapping with soaked absorbent paper for 4 d. The sensitivity of pathogens to five fungicides was determined by the mycelial growth rate method, i.e., the mycelial disk of pathogens with a diameter of 0.5 cm was inoculated on PDA containing different concentrations of fungicides, and the colony diameter was measured by the cross method after 5 d at 25 ℃.【Results】The results showed that the colony of the F. solani root rot pathogen was light pink, with a small amount of white thin aerial hypha growing on PDA for 7 d, and directly produced conidia on PDA. According to the observation on conidia under the microscope, the large conidia were fusiform and obtuse at both ends with 2-4 septums, and the size was (22.5-35.0) μm × (2.5-3.5) μm. The small conidia were oval, with 0-1 septum, and the size was (5.0-18.0) μm × (2.5-3.0) μm. The fragment amplified by the universal primers ef1/ef2 was 735 bp, and BLAST analysis showed that the sequence was 99% identity to F. solani (GenBank accession:KR935896.1). The fragment amplified by the universal primers ITS1/ITS4 was 544 bp, and BLAST analysis showed that the sequence was 97% identity to F. solani (GenBank accession:MK250905.1). Phylogenetic tree analysis based on ITS and tef1- α sequences of F. solani showed that FS-isolates and F. solani clustered together, combined with the morphological characteristics of the strains, and these pathogens were identified as F. solani. The results of host specialization showed that the pathogens could infect watermelon, melon, gourd and pumpkin. The sensitivity of the pathogens to different fungicides was determined. The results indicated that prochloraz had the highest activity inhibiting mycelial growth of the pathogens, with an average median effective concentration (EC50) value of 0.13 mg · L- 1 , followed by carbendazim with an average EC50 of 1.262 mg · L- 1 , while azoxystrobin had the weakest inhibitory effect, and EC50 of all tested strains was greater than 800 mg·L-1 . 【Conclusion】In this study, F. solani causing Cucurbitaceae root rot of watermelon, melon, gourd, pumpkin and other Cucurbitaceaes was identified in Zhejiang. Pathogenicity analysis showed that F. solani could infect a variety of Cucurbitaceae crops without obvious host specialization. The fungicide sensitivity test showed that the three fungicides including prochloraz, carbendazim and iprodione, were effective against F. solani mycelial growth, which could provide theoretical basis for disease control in the field. The F. solani stains in this study can infect watermelon, melon, gourd and pumpkin, so rotation between these crops cannot solve the root rot problem. The purpose of using pumpkin or gourd grafting is to solve the problem of soil-borne diseases. In this study, it was found that pumpkin or gourd grafting rootstock itself would cause root rot, leading to crop failure. At present, there are few studies on the breeding of Cucurbitaceae crops for resistance to F. solani root rot. In the future, it is necessary to breed not only the scion varieties with resistance to F. solani root rot, but also the rootstocks.