Resistance evaluation of 20 pear varieties (germplasms) in China to foreign strains of Erwinia amylovora

Abstract:【Objective】Fire blight, caused by the bacterium Erwinia amylovora, is one of the most destructive disease affecting plants of family Rosaceae. The bacterium can destroy blossoms, shoots and stems, and may seriously damage an entire pear or apple orchard within a growing season under optimal conditions. The disease has currently spread more than 60 countries, and is a serious concern to growers of pears or apples worldwide. Up to now, fire blight and Erwinia amylovora have not yet recorded in China, but have been found in neighbouring countries like Kazakhstan, Kyrgyzstan and Russia during last decade. The control measure consists of removal of diseased host plants or their parts, cultural practices and application of chemical sprays. However, these measures are not always satisfactory. In all the ways, growing relatively resistant cultivars and rootstocks can be one of the most efficient control methods for fire blight. The disease could be better controlled by understanding virulence of Erwinia amylovora strains and responses of host-plant cultivars. The aim of this study was to test pathogenicity of exotic E. amylovora strains and resistance of domestic pear varieties to these strains, so as to provide the foundation for the prevention and treatment of the disease.【Methods】The experiment was conducted in a screen house using in vitro culture assay. The relative virulence of three different E.amylovora strains, E.a 0001 isolated from apple of Kyrgyzstan, E.a 0017 from pear of Kyrgyzstan and E.a 0055 from pear of USA, was examined by both in vitro artificial inoculation twigs and young fruits of testing pear cultivars. The plant materials for testing were obtained from Luntai National Pomology Germplasm Garden, Xinjiang. The shoots collected from healthy pear trees were cut into the sections of 25 cm in length, then sterilized with 75% alcohol, and later 1/3 of the branches were inserted into the triangular bottles containing sterilize-distilled water. Sterilization surgery knife was used to cut a wound of about 5 mm on the shoots surface. On the wound was placed a small degreasing cotton piece saturated with 2 mL tested bacteria suspension at the concentration of approximately 1 × 109 CFU · mL^-1, and then wrapped with plastic wrap to prevent desiccation. The inoculated branches were cultivated in an artificial climate chamber at 28.5 ℃, 75% RH and 12 h light. Twenty shoots from each species were inoculated with each bacterium in the experiment. The length of resulting necrosis was measured 7 days after inoculation, and statistics on the incidence and index were carried out. The virulence of the E. amylovora strains was divided into the strongest, stronger, medium, and weak virulent class according to the disease index. The gathered pear fruitlets were soaked with 75% ethanol for 10 minutes. After the ethanol was volatilized, they were cut in half with sterile scalpel. The sterilization bamboo stick was dipped in bacterial colony to drip on the sarcocarp, and 2 to 4 inoculation points were selected on each section, and the inoculation sterile water was used for negative control. After inoculation, the fruits were put into a sterile Petri dish, which was placed in a germination box with absorbent paper in an incubator. The pathogenicity of the strains was analyzed by recording the appearance time of initial bacterial oozes and the ratio of bacterial oozes area to fruitlet area. Twenty-one pear varieties were chosen for testing their relative resistance to E. amylovora 0017 strain that was aggressive in the above test. Through in vitro shoot culture inoculation method, the level of resistance to E. amylovora was evaluated according to the extent of lesion development on the shoot and the population of the bacteria in the tissue with real-time fluorescence quantitative PCR assay. The level of resistance was converted into six class evaluation scales comprehensively.【Results】The result of pathogenicity test revealed that all strains had different virulence, and were highly strong virulent to the‘Kuerlexiangli'‘Dangshanli'‘Heisuanli'and Pyrus betulifolia, being most virulent to‘Kuerlexiangli'. All strains had strong virulence to‘Xinli No.7', ‘Heisuanli', and‘Hesejujuli', while they had medium virulence to‘UChe Amute'and‘Huochengdonghuangli'. Fruitlets were most sensitive to fire blight and fruitlet inoculation was a common method to identify the pathogenicity of E.amylovora. The results indicated that three tested stains (E.a 0001, E.a0017 and E.a 0055) all had pathogenicity to the measured fruitlets. After inoculation, the bacterial pus generated within 10-24 hours. According to the time of appearance and amount of bacterial pus on the fruitlets, it was inferred that E.a 0017 strain was more virulence to fruitlets than the additional two strains, but it was hard to compare the pathogenicity between E.a 0001 and E.a 0055. The level of resistance of the twenty-one pear varieties to E.amylovora wasn't higher generally. The majority of the pear cultivars tested was susceptible to E.amylovora, 14 pear cultivars (70%) were evaluated as varying degrees susceptible, including 4 highly susceptible, 7 susceptible and 3 moderately susceptible cultivars.None of them was highly resistant to the pathogen, while 2 cultivars performed as resistant, and 4 cultivars as disease-tolerant, which were all local varieties in our country.【Conclusion】A system for testing virulence and host plant resistance to Erwinia amylovora have been developed in laboratory. The variability in virulence among the strains was found, with E.a 0001> E.a 0055> E.a 0017. Evaluation of obtained results proved the varieties with a higher level of resistance were‘Lüli'and‘Jinsu'.‘Bayuesu', ‘Huochengdonghuangli'and‘UChe Amute'and‘Mianli'were evaluated as disease-tolerant.‘Huangsuanli'‘Jinchuanxue'and‘Hesejujuli'were evaluated as moderately susceptible.‘Xuehuali'‘Pyrus Betulifolia'‘Hongxiangsu'‘Qipan'‘Hongxiangli'‘Zaosu'and ‘Xinli No.7' were evaluated as susceptible.‘Kuerlexiangli'‘Dangshanli'‘Jinhuali'and‘Huangsuanli'were evaluated as highly susceptible.