Isolation And Iditification Of The Jujube Shrink Fruit Disease Pathogens And Diversity Analysis Of Endophytic Fungi In Jujube

In view of the serious question about this disease, this research have studied the symptoms characteristics of this disease in many jujube planting areas of Xinzheng Henan province, We identified the pathogens by pathogenicity testing, culture features as well as morphological and molecular characters. Also we first found a new bacterial pathogen and identified two fungal pathogenic speciese of Fusicoccum and Phoma. This research also have first studied the diversity of endophytic fungi in Jujube. The results are as below.1. Disease symptoms characteristics and isolation of pathogens:At the beginning stage of the disease, there were yellow irregular spots on the shoulder or the middle part of the fruits; then the area of the sports grown larger and larger, the color of spots become brown and dark brown.At the same time the spots sink slightly.Later, the spots become more and more wrinkled until wizened.The sarcocarp diseased look like sponge and taste bitter greatly. 266 strains were isolated from 201 disease fruits which were collected from Xinzheng and Neihuang by regular isolation methods during 2008-2009. According to the morphological characters and culture features, they were initially classified as five fungal species (strains zs081, zs082, zs083, zs091and zs092) and two bacterial species of possible pathogens. Overall, strains zs082, zs083, zs091 had high separation frequency.2. The five fungal species and two bacterial species are inoculated in lab and field. Based on the pathogenicity experiment the results showed that strains of zs082, zs083, zs091 and zs09b2 can lead to disease. They showed the same symptoms as in the field. The pathogens isolated from the disease fruits is same as the strains of inoculation. These results proved that the strains zs082, zs083, zs091 and zs09b2 were the pathogens of Jujube fruit shrinking disease.3. According to the cultural characteristics, morphology and physiological and biochemical characteristics, zs082 was identified as Fusicoccum aesculi Corda, zs083 was identified as Phoma glomerata (Cda) Wollenw. zs091 was identified as Alternaria alternata(Fr.)Keissler, zs09b2 was identified as Bacillus amyloliquefaciens .4. The three pathogens: zs082, zs083, zs091, were cloned and sequenced using the universal primers ITS1 and ITS4, and were compared with published sequences in GenBank database. The results showed that Botryosphaeria dothidea. were 100% identical to zs082. However, the sexual generation of the F. aesculi is B. dothidea, this was consistent with the morphological identification results. P. glomerata were 100% identical to zs083. And zs083 has the same morphology characteristic with P. glomerata. So we identified it as P. glomerata. The strain zs091 had 100% identical to Alternaria tenuissima and A. alternate. But A. alternate had the same morphology characteristic with zs091.In order to defined the classification status of zs082 again, we detected the elongation factor 1-a (EF1- a) gene sequence of it. The result indicated that the sequence of it has 99% homology with B. dothidea. And this was the same as the result of ITS sequences blast.16S rDNA sequence of pathogenic bacterium zs09b2 has 99% homology with Bacillus sp. The 16-23s ITS rDNA sequences has 100% homology with B. amyloliquefaciens.5. In order to study the species and distribution of the endophytic fungi in Jujube, using regular isolation metheds, 1511 strains of endophytic fungi were isolated from barks, wattles, branches, leaves, blooms and fruits which in whole growth period and various varieties of Jujube with the routine isolating method. These strains were classified into 15 genera, which showed higher diversity of endophytic fungi in Jujube. Alternaria, Phoma, Fusarium and Nigrospora, are dominant genus. It was found that the amount, species and distribution of endophytic fungi varied in different growth periods and tissues of Jujube.Through identifying the endophytic Alternaria from different periods and tissues and varieties of Jujube, we found the dominant species was Alternaria alternata (Fr.) Keissler. It is the same as the pathogenic species. We initially determined that the pathogenic Alternaria possibility had long time latency features in Jujube.