Abstract: 【Objective】 The study was first of all to determine some plant hosts of A. alternata existing in pear trees orchard. Then the plant hosts could be caused diseases by A. alternata from pear leaves with typical black spot disease. At last, A. alternata from these diseased plant hosts has ability of pathogenicity on pear leaves. The research attempted to analyse plant hosts of A. alternata existing in pear orchards, and then these plant hosts containing A. alternata may be one of the potential infection sources for pear black spot disease.【Methods】 A. alternata, which was stored in -4 ℃, was firstly activation and cultivation using PDA agar medium at 26 °C±1 °C for 4 days. The activation 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 for comparison in follow-up experiment. Healthy pear leaves were inoculated with activation A. alternata suspension to demonstrate A. alternata pathogenicity on pear leaves. For 8 days later, we would observe 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 °C± 1 °C in the dark for 72 h. This step was for A. alternata isolation. A microbe was isolated from the diseased pear leaf. The white mycelium of the microbe were got from PDA agar medium by sterilized scissors, 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 trees orchard, twenty-five different kinds of plant species were provided to inoculate the same volume of A. alternata isolation suspension respectively. All plant species were transplanted into a incubator for 16 h at 28 °C (day) and 8 h at 25 °C (night). Relative humidity in the incubator was 80% ± 5%. All treated samples were for incubation in the incubator for 14 days. According to leaves symptoms and lesion areas of statistical analysis, leaves with obvious symptoms were for pathogen isolation again. The purification second generation of pathogen from diseased leaves as to A. alternata were observed. In order to identify the second generation of pathogen as to A. alternata has 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 would observe 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 observed 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 has the same phenotype as to A. alternata. A. alternata isolation was identified by method of nested PCR, the same bands as prediction were 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 were 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, 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 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 were 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 were widely existed in pear trees orchard. A. alternata from these plant hosts has ability of pathogenicity on pear leaves. It indicated that plant hosts containing A. alternata may be one of the potential infection sources for pear black spot disease epidemic.
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