Identification of the pathogens of blossom blight and screening of fungicides (bactericides) in Guichang kiwifruit

Abstract:【Objective】The disease damages kiwifruit buds, which have been found in Xiuwen county of Guizhou province. It causes the calyx to show dark brown, the filaments and anthers to become rot,which is called blossom blight of kiwifruit. It has so significant effect on the opening and pollination of the flower buds that it can result in a decline in the fruiting set rate and economic losses. It is important to determine the pathogen of Guichang kiwifruit flower rot. To screen out fungicides (or bactericides)that possess high-efficiency and low-toxic properties for prevention and control for blossom blight of ki-wifruit, the experiment was carried out, so that the economic loss caused by the disease could be re-duced.【Methods】The diseased symptomatic tissues were surface-disinfected with 75% ethanol for 3-4s and rinsed three times in sterile distilled water, and then cultured on the beef extract peptone medium (NA) with 75% relative humidity at 25 ℃. The isolated pathogens were tested for the pathogenicity of kiwifruit buds by using the local inoculation method, and the pathogenicity of the pathogens to Nicotia-na benthamiana, Lycopersicon esculentum and Apium graveolens L. were tested with another method.The morphology of the colony on NA medium was observed, after Gram staining, capsule staining, spore observation, and scanning electron microscopy as the basis for morphology. LOPAT test was un-dertaken by gelatin liquefaction, starch hydrolysis, glucose oxidation fermentation, malonic acid utiliza-tion, citrate utilization, esculin hydrolysis, hydrogen peroxide and phenylalanine deaminase, fluores-cence, methyl red and V-P test, whether it can use sorbitol, mannitol, glucose, ribotide, erythritol and su-crose, acting as a strong basis for researches on physiological and biochemical characteristics. The PCR amplification used16S ribosomal DNA, DNA replication initiation protein (dnaA), DNA gyrase B sub-unit (gyrB), RNA polymerase sigma-70 factor (rpoD) and citrate synthase gene (gltA) primers. NCBI’s BLASTn tools were used to obtain highly homologous DNA sequence in GenBank and molecular phy-logenetic tree was constructed by MEGA 7.0.14. The bacteriostatic zone method was used to screen the virulence of 11 kinds of fungicides (bactericides) and their combination against blossom blight of kiwi-fruit, so as to obtaine the related toxicity regression equation.【Results】Four days after inoculation, arti-ficially infected flowers showed symptoms alike those observed in the farm, whereas the control was as-ymptomatic. After the same measurement was used, which was described above with the original strains, the bacteria were isolated and purified from the infected tissues. Inversely, it was not isolated from the control, which fully conformed to the verification of Koch’s Postulates. After the fruits, stems and leaves were infected by Nicotiana benthamiana, Lycopersicon esculentum and Apium graveolens L., the necrotic spots and pus produced. The pathogen was off-white, smooth and translucent with neat edges on NA medium. It was a gram-negative bacteria with the size of (2.32-1.77) μm× (0.497-0.663) μm.The strain belonging to the LOPAT II group (- - + - +, L negative, O negative, P variable, A negative, and T positive) did not produce spores and had capsule. It was capable of producing fluorescent pig-ments, causing fermentation of glucose, using citrate and malonic acid, and slightly liquefying gelatin and hydrolyzing esculin. However, it could not hydrolyze starch. Catalase peroxide was positive and phenylalanine deaminase was negative. The results of methyl red and V-P showed that the former was positive and the latter was negative. It can utilize glucose, sucrose, erythritol and sorbitol as carbon source except ribonic acid and mannitol. BLAST on the NCBI official website was used for analysis of the sequence that used universal primer 27F/1492R. The results showed that strain G-2 (GenBank acces-sions: MT950156) and Pseudomonas viridiflava (GenBank accessions: AY180972.1) were 100%, Pseu-domonas graminis were out-of-group strains in the periphery. The primers rpoD-FP/RP, gyrB-F/R, ctsF/R and M209 F/R were used for PCR amplification and comparative analysis of strain G-2. The phyloge-netic tree showed that strain G- 2 (GenBank accessions: rpoD, MT975512; gyrB, MT994325; gltA, MT975511; dnaA, MT975513) cannot be distinguished from other sources of Pseudomonas viridiflava, but they were all in the same group. The susceptibility of the bacteria to bactericides (fungicides) showed that tetramycin had the highest antibacterial activity against the pathogen among the 11 kinds of fungicides, with an EC50 of 1.24 mg·kg^-1, followed by prothioazole with an EC50 of 9.62 mg·kg^-1. Both bactericides (fungicides) were combined, the CTC values were 272.70 and 129.86 when the effective mass ratio was 4∶1 and 3∶1. This result suggested that it had a synergistic effect.【Conclusion】It was proved that the pathogen of blossom blight of kiwifruit in Guizhou province is P. viridiflava through morphology, combined with biological characteristics and molecular biological methods. Tetramycin, prothiazole and other effective prevention and control fungicides (bactericides) and combination agents were screened out by the test. But the final control effect of the agent still needs to be verified in the field. This study provides a theoretical basis for the development of rapid detection technology that pre-vents blossom blight of kiwifruit in advance.