| Litchi chinensis is a famous subtropical fruit in the southern China, also is one of the most intolerant storage fruit. Pathogen infection and fruit browning were the main reasons affecting in its storage period. Presently, chemicals were commonly used in controlling this disease. But there are more shortcomings, such as pesticide residues, environmental pollution and so on. So that biological control has became the hotspot study. Otherwise, the vegetative of Oomycete is diploid. Traditional method of gene mutation such as knockout was not feasible to use in Oomycetes. Transforming endogenesis gene and causing its silence are the most technique to study the gene function of Oomycetes. In present, there is a lack of efficient molecule genetic manipulation, and the study on molecule level is so slow. Therefore, the Biological characteristics of lychee post-harvest disease pathogens, transformation system, the control or preservation by endophytic bacteria were systemicly studied in this paper. The main rusults are as follows.Biological characteristics of Peronophthora litchii. The biological characteristics of P. litchii demonstrated that the growth of its sporangiophore belongs to multi-determinative type. The sporangia could germinate to form various shapes, zoospores, tubes, sporangiophore, or secondary sporangia. The germinating holes might locate in the one side, the both sides or roundness of the sporangia. In one hole one or more radical buds might germinate out. The base holes of the sporangia usually are easier to germinate and could form chlamydospores and oospores after culturing for 35 and 45 d respectively. The sporangia produced from the fungus cultured for 10 d would have the best germinating rate. The sporangia germinated to form zoospores at 16 ~ 24℃and tubes at 26℃. The more spores were produced on PDA plate, and the fungus grows fastest on the oat plate. The most suitable pH for its growth is 6~8; In the same time, The influencing factors on the mycelial growth, sporulation, sporangia germination and infection of P. litchtii were systemicly studied. The result showed that the optimum conditions for the mycelia growth and sporulation of P. litchii were in the medium of pH 7.0 and at 24℃. The optimum illumination for the mycelial growth and sporulation of the fungus were under light for 20 h and 16h separately. The lethal temperature of the sporangia was 45℃, keeping for 10 min. The results also showed that the germination form and rate of sporangia were different when they in various temperatures, illumination, pH values and nutrient compositions. The best germination rate of the sporangia was from the sporangia which were produced from the fungus cultured on PDA medium (pH7.0) at 24℃and under light for 20 h . And the disease was the most serious when the fungus infested the lychee fruit at 24 ~ 28℃. The ultra-structures of the fungus showed that it could invade lychee fruit peels through the lenticels and nods with sporangia or zoospores, then germinate and spread under lychee pericarp.Biological characteristics of Geotrichum candidum. The result showed that the optimal medium for the fungus growth and sporulation was PDA. The fungus grows the slowest on the chaff plate and the fewest spores were produced. For the mycelial growth and sporulation of G. candidum, the optimum growth conditions were from 20 to 28℃, pH 6 ~ 9 and 12 ~ 20 h illumination. The lethal temperature to conidia was 65℃. In addition, the effects of pH, medium, illumination time, temperature and nutrients on the conidial germination of the fungus were studied. The results showed that when the temperature was 24 ~ 32℃and the illumination time was 20 h it had the highest germination rate. The optimum temperature for their infection was from 28 ~ 32℃.Apart from lychee, the pathogen could infect carrots, tomatoes, peaches, longan, grapes, eggplant and cucumber as it was artificialy inoculated.Construction of P. litchii’s transformation system. The best conditions for protoplast formation of P. litchii were as follows, the mycelia age was 4d,protoplasts of osmotica were produced by digesting the cell wall for 4 h at 30℃with 15 mg·mL-1solution of driselase, regeneration was in the LBA medium applied with 0.6 mol·L-1. Regenerated strains grew on the LBA medium, forming gray,orbicular and felty colonies, and showed pathogenicity to the host plant similarly as did their parent.The gfp gene was introduced into the P. litchii strain by pHAM35G, the Brema lactucae expressing vector ham34. However there was no green fluorescence observed by Olymplus microscope.Construction of G. candidum’s transformation system. The best conditions for the protoplast formation of G. candidum was as follows, the mycelia age was 1d,the protoplasts of osmotica were produced by digesting the cell wall for 4 h at 30℃with 5 mg·mL-1solution of driselase, regeneration was in the YEPD medium applied with sucrose (20%). The colonies of the regenerated strains were white, orbicular and the surfaces were powdery. The strains also showed pathogenicity to the host plant similarly as did their parents.The gfp gene was introduced into the G. candidum strain by pHAM35G, the Brema lactucae expressing vector ham34. And there was green fluorescence observed by Olymplus microscope.Screening of the antagonistic and endophytic bacteria against P. litchii. Two hundred and ten bacterial strains were isolated from plants. Among them one hundred and fifty five endophytic bacterial strains were found inhibit fungi P. litchii and G. candidum, accounting for 73.81% of the total isolates. Nine strains were the better-performing strains, as their antagonistic bandwidth is greater than 10μm, accounting for 5.81%. And, the test results showed that among the antagonistic strains, Bacillus subtilis BS-2, TL2 and B. amyloliquefaciens TB2 had obviously better inhibition to the fungi P. litchii and G. candidum, and could delay the browning speed of lychee pericarp.Colonization of endophytic bacterial strains in side of lychee. The strains BS-2 and TB2 were mutanted by antibiotics and labeled by gfp gene, then inoculated by daubing leaves, flower, younger fruit and older fruit. The two strains were found to colonize in the tissue of lychee, and they were also able to live and move in the flower to the younger fruit of lychee. Moreover, with the changes of the seasons and the plant growth cycle, the colonization situation of the endophytic bacteria in lychee leaves would also be changed, such as the colonization time in leaves significantly longer in spring than that in fall. The colonization conditions were also different in the different tissue sites of the host. So that, the two strains could colonize in the epidermal cells of lychee leaves, palisade tissue and intraepidermal cells, and in the epidermal cells of lychee fruit, palisade tissue and endocarp. The results also showed that the colonization quantity of the bacterial strains had a positive correlation with control effect and preservation effect.Control effect of post-harvest disease by endophytic B. subtilis strains and their control mechanisms. The two endophytic bacterial strains were used to control post-harvest disease of lychee. The results demonstrated that the strains TB2 and BS-2 had a good control effect on the post-harvest disease of lychee. The control effect were 61% ~ 96.01% and 33.73% ~ 90.86 % in 6d after inoculation with the two strains. The research result of biological control mechanisms showed that the endophytic bacteria could inhibit the mycelial growth and reduce the sporangia or conidia formation and germination of P. litchii and G. candidum. The endophytic bacteria could be increased the activities of SOD, CAT,β-1, 3 -glucanase and chitinase, and the content of glutathione(GSH) and ascorbic acid (ASA). But the activities of peroxidase (POD) and polyphenol oxidase (PPO), the content of MDA, the rate of O2- were lower.Preservation effect of lychee by endophytic B.subtilis strains and their chemical mechanisms. The results showed that the strains BS-2 and TB2 could reduce the different breed lychee fruit peel from browning. After inoculation with the two endophytic bacterial strins, the healthy fruit rate were 246.81%~248.89%(Dongliu 1)and 38.35%(Yuanhong)higher separately than control. The main control mechanisms could be that the endophytic bacteria could increase the activity of SOD and CAT, decrease the content of MDA and the rate of O2-. In the same time, the endophytic bacteria could decrease the activity of PPO, POD and anthocyanase which could decrease the content of phenolic and reduce the degradation of anthocyanin. And more, the strains BS-2 and TB2 could increase sugar, Vc and organic acid content. In addition, the total amount of amino acids in the lychee kept the same as the control in 7d after treating with the bacterial strains. |
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