| Mulberry fruit sclerote disease is also called swollen fruit disease. According to the various of pathogens and symptom, mulberry fruit sclerote disease are divided into three kinds, which are the mulberry sorosus hypertrophic sclerote disease, mulberry sorosus diminuting sclerote disease and mulberry sorosus parvulling sclerote disease. Although they have different pathogens, they share the same infection cycle. At present, these disease occures almost all over the country and causes great harm to mulberry fruit industry. And, controlling of the diseases is mainly relying on chemical and agronomic measures. However, the effect of agronomic measures is limited.. Excessive use of chemicals brings many problems, such as pesticide residues, environmental pollution, threating mulberry sericulture and edible safety. Therefore, it is extremely important to find safe, efficient and environmentally friendly biological control methods.Endophyte is widely distributed in terrestrial and aquatic plants in vivo, and inhabitating in plants. As a new biological control agent, endophytes are described as microorganisms that colonize the tissues of healthy plants without causing apparent pathological symptoms and establish a harmonious relationship with host plants. They can improve resisitance of the host through producing antibiotic, inducing systemic resistance and promoting plant growth. Because of less affected by environmental conditions, endophytes can colonize, spread and pla y a role in biocontrol for a long time. Therefore endophytes are becoming a potential biocontrol resource and receiving much attention.The pathogens of mulberry sorosus hypertrophic sclerote disease and mulberry sorosus diminuting sclerote disease are isolated from diseased mulberry fruits and apothecias. Then morphology and molecular biology methods are used to identify them. Moreover we studied their cultural characteristics. Using two pathogens of mulberry fruit sclerote disease(obtained in present research) as the target, one endophyte was screened out, which can significantly inhibit the growth of the target pathogens and promote the germination and growth of mulberry seedings. Then preliminary research was carried on to reveal growth-promoting mechanism. All of the study laid the foundation for the biological control of mulberry fruit sclerote disease. The main results of the research are as follows. 1 Isolation and identification of pathogens of mulberry fruit sclerote disease and research on their culture characteristics101 isolates were isolated from diseased mulberry fruits and apothecia by surface disinfection. Among them, 12 isolates were identified to Helotiales, Geoglossaceae, Scleromitula, Scleromitula shiraiana based on the phylogenetic analysis of ITS and morphological features. The isolate of Sclerom itula shiraiana SXSG-5 was inoculated to the mulberry flower in field, and the results showed that mulberry fruits vaccinated with mycelia of SXSG-5 strain became diseased and showed the symp tom of mulberry sorosus diminuting sclerote disease. And then, Scleromitula shiraiana could be isolated from the diseased mulberry fruits again. Then we studied the culture characteristics of it. The results showed that the SXSG-5 strain couid grow in different mediums, and it could grow faster on PDA and form a lot of mycelia on mulberry leaf juice of potato dextrose medium. The optimum growth temperature of SXSG-5 was 20℃, the optimum growth p H was 6.0. Light conditions could affect the growth of SXSG-5, which growed faster in full light than 12 h lighting 12 h dark, and growed slowest in dark condition.27 isolates of pathogens from club-shaped apothecia were isolated by surface disinfection. Among them, 11 isolates were identified to Ascomycotina, Leotiomycetes, Helotiales, Sclerotiniaceae, Sclerotinia, Sclerotinia sclerotiorum based on the phylogenetic analysis of ITS and morphological features. The isolate of Sclerotinia sclerotiorum PZ-2 was inoculated to the mulberry flower in field, and the results showed that mulberry fruits vaccinated with mycelia of PZ-2 strain became diseased and showed the symptom of mulberry sorosus hypertrophic sclerote disease. And then, the Sclerotinia sclerotiorum could be isolated from the diseased mulberry fruits by tissue isolation again. Then its cultural characteristics were studied. The results showed that the PZ-2 strain could grow in different mediums, and it could grow faster on PDA, NA and mulberry leaf juice of potato dextrose medium. The optimum growth temperature of PZ-2 was 20℃ to 25℃, p H and light conditions could not affect the growth of PZ-2. 2 Isolation and identification on mulberry endophytic antagonistic fungi and its culture characteristics.Using pathogens of Scleromitula shiraiana SXSG-5 and Sclerotinia sclerotiorum PZ-2 as target, one antagonistic endophyte of mulberry stem SAJ-1 strain was screened out. Then it was identified by the molecular biology and morphological features.The results are as follows, three weeks culture of SAJ-1 strain can inhibited the growth of Scleromitula shiraiana SXSG-5 and Sclerotinia sclerotiorum, and the inhibitory rates were 98.07% and 93.42% respectively. Moreover, it displayed a stable inhibitory effect on them. Powdery and gray solids formed on PDA after inoculation 12 weeks, which was identified as pycnidia under a microscope. Besides, the pycnidia formed on WA and OA for 12 weeks. The pycnidia were olivanceous brown to b lack and globose or flask shaped, 98.0~126.9 μm×158.3~186.0 μm.The pycnidial was of textura angularis septate setae, 30.8~93.1 μm distributed on the surface of the pycnidial wall. Conidiogenous cells arosed from the entire inner surface of the pycnidial wall, 50.0~80.0 μm×110.0~268.0 μm. Condia were whitish in mass, ellipsoid, hyaline, 0.8~1.5 μm×2.3~3.1 μm. Based on the phylogenetic analysis of 28 S r DNA gene sequences the SAJ-1 strain closed to Pyrenochaeta unguis-hominis(GQ387621) and within the minimal clade. Based on the phylogenetic analysis of ITS gene sequences SAJ-1 strain and Pyrenochaeta sp.(EU885415) within the minimal clade. Meanwhile, compared the morphological characteristics of the strain SAJ-1 with Pyrenochaeta unguis-hominis, we fonund they were differnt in shape, size, colony morphology on OA medium and internal structure of the condia. Therefore, based on the phylogenetic analysis of ITS and morphological observation, the SAJ-1 strain belonged to Pyrenochaeta sp., and was named Pyrenochaeta.sp. SAJ-1.Inhibitory spectrum test results showed that 15 target pathogens such as Ceratocystis ulmi, Botrytis cinerea, Verticillium dahlia, Cercospora beticola, could be inhibied by SAJ-1 strain. Then the cultural characteristics of it showed that the SAJ-1 strain could grow in different mediums, which could grow quickly on O A, CZA and WA. The optimum growth temperature was 25℃. It grows good in three kinds of light conditions 3 Effect of entophytic fungi strain SAJ-1 on seed germination and seedling growth of mulberry.Pyrenochaeta sp. SAJ-1 had a good antagonistic on the pathogens of mulberry fruit sclerote disease, However, the possibility of applying this strain to the biological control of mulberry fruit sclerote disease in field, still needed to explore their impact on the growth of mulberry. So the influence of Pyrenochaeta sp. SAJ-1 of different dilutions of mycelium suspension and cultural filtrate on seed germination and seedling of mulberry growth was studied by greenhouse experiments.Seed germination experiment results revealed that the seed germination ability and germination rate of mulberry seed were higher than the control group when treated them with 100-fold and 500-fold dilution of mycelium suspension. The seed germination ability were increased 40.54% and 24.32% respectively; the germination rate were increased 23.91% and 15.21%. This results also indicated that 100-fold dilution and 500-fold dilution of mycelium suspension can promote mulberry seed germination. The seed germination ability and germination rate of mulberry seed were lower than the control group when treated them with 0- fold dilution, 100- fold dilution and 500- fold dilution of culture filtrate. This results indicated they inhibited mulberry seed germination. While the seed germination rate treating with 10-fold dilution of culture filtrate almost same as the group dealing with sterile water.Mulberry seeding growth-promoting test results showed different dilution multiple of fungi mycelium suspension could increase the number of apical roots per plant, fresh weight of root part, the plant fresh weight, the plant height and the fresh weight of aerial part. Among them, the group treated with 10-fold dilutions of mycelium suspension was the best. Compared to the control treated of sterile water, the number of apical roots per plant, the length of taproot, the fresh weight of root part, the plant fresh wight, the plant height and the fresh weight of aerial part increased 50.00%, 27.92%, 79.87%, 112.76%, 22.28% and 141.71% respectively. 0- fold dilution, 10- fold dilution, 100-fold dilution and 500- fold dilution of culture filtrate could increase fresh weight of root, the plant height and the fresh weight of aerial part. Among them, the group treated with 10- fold dilutions of culture filtrate was the best. Compared to the control treated with sterile water, the length of taproot, the fresh weight of root part, the plant fresh wight, the leaf blade length, the width of leaf blade aerial part, the plant height and the fresh weight of aerial part increased 3.33%、139.60%, 77.89%, 26.45%, 52.50%, 20.27% and 60.30% respectively. Further results showed that that SAJ-1 strain could promote the growth of mulberry by producing IAA and dissolving the calcium phosphate.Test results showed that SAJ-1 strain the capability of producing IAA increased in the first 12 days, and then stabilized as time goes on. The peak amount of IAA was 13.16 μg/m L, when it cultured for 12 days. In addition, a diameter of 24.83 mm transparent ring formed on the inorganic phosphorus plate inoculated the SAJ-1 strain. The maximum of dissolving phosphorus was 447.91 μg/m L for 7 days after inoculation. Hereafter, the amount of dissolving phosphorus began to decrease and stabilized finally.In summary, the pathogen Scleromitula shiraiana SXSG-5 of Mulberry sorosus diminuting sclerote disease and Sclerotinia sclerotiorum PZ-2 of Mulberry sorosus hypertrophic sclerote disease were isolated and identified. And then, using the two pathogens as targets, one antagonisitic endophyte was screened and identified. Furthermore, this strain was not only no negative impact on the growth of mulberr y, but also couid promote seed germination and seedling growth of mulberry. All of the above reseach provided the materials and experimental basis to the biological control of mulberry fruit sclerote disease. |
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