Analysis of plant species enabling to infect with Alternaria alternata in pear orchards

【Objective】The study aimed to determine some plant hosts by Alternaria alternata in pear orchards and these plant hosts could show typical black spot disease. Simultaniously, A. alternata from these diseased plant hosts has the pathogenicity on pear leaves.【Methods】The pathogen A. alternata stored at -4 ℃, was activated and cultivated using PDA agar medium at (26±1) ℃ for 4 days. The activation of A. alternata was identified by nested PCR for containing ITS gene of A. alternata. The nested PCR results on ITS gene of A. alternata were used for comparison in follow- up experiment. Healthy pear leaves were inoculated with activated A. alternata suspension to demonstrate its pathogenicity on pear leaves. For 8 days later, we observed symptoms on pear leaves. Nest PCR reaction on the ITS gene of A. alternata was used to detect the diseased pear leaves. The diseased pear leaf tissue of 1 cm2 was inoculated on the PDA agar medium at 26± 1 ℃ in the dark for 72 h. This step was carried out for A. alternata isolation. A microbe was isolated from the diseased pear leaf. The white mycelium of the microbe was gotten from PDA agar medium with a pair of sterilized scissors, and then were inoculated on a new PDA agar medium 96 h for purification. The appearances of colonies were observed. The colonies were identified by nest PCR reaction on the ITS gene of A. alternata. In order to determine plant hosts of A. alternata existing in pear orchards, twenty-five different kinds of plant species were provided to inoculate the same volume of A. alternata isolation suspension respectively. All plant species were transplant-ed into a incubator for 16 h at 28 ℃ (day) and 8 h at 25 ℃ (night). Relative humidity in the incubator was controlled to 80% ± 5%. All treated samples were incubated in the incubator for 14 days. According to leaf symptoms and lesion areas, leaves with obvious symptoms were used for pathogen isolation again. The second generation purification of pathogen from diseased leaves infected with A. alternata were observed. In order to identify the second generation of pathogen, the A. alternata was proved to have the ability of pathogenicity on pear leaves. These second generation of pathogen suspensions were separately inoculated on healthy pear leaves. These pear leaves were placed in another incubator with the same environmental conditions as above. For 10 days later, we observed the symptoms on the inoculated pear leaves. The lesion areas on pear leaves with A. alternata treatment were respectively used for statistic analysis.【Results】The stored A. alternata could be cultured on the PDA agar medium. By nested PCR on ITS gene of A. alternata, we could observe positive bands (570 bp and 398 bp) as prediction. Healthy pear leaves inoculated with A. alternata suspension showed obvious symptoms as to black spot disease for 8 days later. And also, bands as to ITS genes could be detected in the diseased pear leaves by nest PCR. As predicted, the pathogen isolated from pear leaves had the same phenotype as to A. alternata. A. alternata isolation was identified by method of nested PCR, and the same bands as prediction appeared. The results indicated that A. alternata, stored in the laboratory, could also cause pear black spot. According to symptoms on twenty-five different kinds of plant leaves, we discovered the obvious symptoms appeared on some plant leaves, such as apple, Malus spectabilis, cherry, Chinese rose, peanut and jujube. Through statistical analysis of the lesion areas on these six kinds of diseased plant leaves, the significant difference was discovered. We deduced the six kinds of plant species such as apple, Malus spectabilis, cherry, Chinese rose, peanut and jujube were the appropriate hosts of A. alternata. We could isolate six microbes from these six different kinds of diseased plant leaves respectively. All of the six colonies had the same performance as to A. alternata from pear leaves. The healthy pear leaves were inoculated with pathogen suspension from six different kinds plant host leaves respectively. After 10 days, all healthy pear leaves with pathogen suspension treatment showed typical symptoms as to pear black spot. These pear leaves were collected for molecular detection by method of nest PCR on ITS gene of A. alternata. By agarose gel electrophoresis, it showed that same bands as prediction appeared. Results indicated A. alternata from hosts with A. alternata infection could cause diseases on pear leaves.【Conclusion】We discovered some plant hosts of A. alternata widely existed in pear orchards. A. alternata from these plant hosts had the pathogenicity on pear leaves. It indicated that plant hosts containing A. alternata may be one of the potential infective sources for pear black spot disease epidemic.