Approach To Biological Control Of Oil-tea Camellia Anthracnose

The oil-tea camellia anthracnose(main pathogen Colletotrichum gloeosporioides)is the most considerable disease in Camellia oleifera planting areas in Hunan province,which is an important factor affecting the yield and quality of camellia oil.The pathogen of oil-tea camellia anthracnose can infect the flower bud,leaf bud,fruit,branch tip and leaf of C.oleifera.In Hunan province,the average fruit drop rate caused by the pathogen is 20%,sometimes up to 40%-50%.At present,this disease is still prevented and controlled by chemical treating,combined with other cultivation proctices such as garden cleaning and tending.However,this disease has the characteristics of long incubation period and rapid spread,so that it makes difficult to grasp the timing of chemical control.With the growing concern about food safety,the use of chemical-sourced pesticides has been gradually restricted,and people are beginning to look for other safer and more reliable ways.In order to achieve this,we collected C.oleifera sources from all over Hunan province,and searched for effective ways of biological control of oil-tea camellia anthracnose from endophytic fungi,rhizosphere bacteria and fungal viruses.The results are listed below:1.Isolation of endophytic fungi in camellia oleifera and screening of resistance to oil-tea camellia anthracnose.Eighty-one strains of endophytic fungus were isolated from C.oleifera tissues collected from C.oleifera planting areas in our province.The endophytic fungus identified by morphology and molecular biology mainly as,Hemimascomycetes and Pyrenomycetes in Ascomycota.Simpson diversity index analysis showed that the species diversity of endophytic fungus in leaves was the highest,followed by in bark,and the lowest in fruit.The antagonistic effect of endophytic fungus against C.gloeosporioides was screened by plate confrontation method,but no strain with obvious antagonistic effect was found.(This section is published in Mycobioloy 2018,46(2):85-91,SCI,IF=1.369)2.Screening,action mechanism and field application of antagonistic bacteria against oil-tea camellia anthracnose.(1)Screening of bacterial strains.Twenty-three strains were isolated from the rhizosphere soil of Camellia oleifera.Among those,P-14 had the strongest inhibition effect,with inhibition zone diameter up to 26 mm.By means of biochemical characteristics and 16 S rRNA gene sequence analysis,P-14 was identified as Bacillus amyloliquefaciens?In vitro experiment showed that the control effect was 100% and 96.08% by soaking detached leaves in fermentation stock and 5-time diluent before inoculation,which was better than foliage spraying of fermentation broth after inoculation.The pot experiment in greenhouse showed that P-14 fermentation broth can effectively control C.gloeosporioides,and 5-time diluent spraying had the best result(77.8%).(2)Antibacterial substances and antagonistic mechanism of P-14.The antagonistic substances in P-14 fermentation broth were separated by acid precipitation and high performance liquid chromatography,and the antagonistic component A(mainly C15 bacillomycin D)was obtained.Volatile substances from P-14 by headspace solid phase micro-extraction and gas chromatography-mass spectrometry showed that the volatile substances could antagonize against C.gloeosporioides,and 35 kinds of volatile substances were isolated.The morphological changes of mycelia treated with P-14 active substance were observed using normal mycelia as a control under optical microscope,scanning electron microscope and transmission electron microscope.It was found that these inhibitors could dissolve the cell wall of C.gloeosporioides and cause protoplasma leakage,resulting in mycelium rupture and spore germination inhibiting.(This section is published in Forest Research 2019,32(1):118-124,IF=1.236)(3)Fermentation conditions optimization and field control technology of strain P-14.The results showed that the optimum fermentation medium for strain P-14 was 0.5% yeast extract,3.0% glucose,0.1% magnesium sulfate and 0.3% sodium chloride.The optimum fermentation conditions were a 250 m L flask with 90 m L liquid,6% of inoculum volume of seed liquid,pH 6.0-6.5,temperature 28 ?,48 h of culture time and shaking table rotation speed 130 r/min.The control effect in mature C.oleifera forest was significantly improved by foliar spraying P-14 fermentation combined with plantation clearance.The control effect was 66.15% only spray in April and 70.52% two sprays in April and June.Spraying P-14 fermentation and foliar fertilizer on young and middle-aged C.oleifera plantations and root fertilization could not only significantly reduce the incidence rate(only 6.32%,lower than 28.08% of reference),but also promote the growth and increase the yield.The average fruit yield per plant was 5.13 kg,2.5 times as more as that of the control,the average plant height was 2.01 m,1.37 times as high as that of the control,and the average crown width was 2.32 m,1.34 times as wide as that of the control.3.Screening and identification of mycovirus from oil-tea camellia anthracnose.Twenty-three strains of C.gloeosporioides were screened for fungal viruses.Only from YZ-2 and YD4-2 strains,mycovirus dsRNA bands were isolated.Two viral bands of 1770 and 1615 bp in size were isolated from strain YD4-2.They constitute a viral genome,named colletotrichum gloeosporioides partitivirus 1(CgPV1).CgPV1 was identified as a new virus of Partitiviridae family by sequence analysis.This is the first time that fungal virus was isolated from oil-tea camellia strains of anthracnose.