| Postharvest fruits rot is a global problem.Apple is an important fruit worldwide.Apple postharvest anthracnose(bitter rot) caused by Colletotrichum gloeosporioides is a destructive disease of apple fruit in production areas;it can cause serious yield loss.In this paper,the techniques of physical,chemical, biological,cultural control and molecular diagnosis for apple anthracnose were used to assembly a sustainable apple bitter rot control system,with aim at ecological environment protection and sustainable agriculture development.The effect and affecting mechanism to apple bitter rot of chemical control,the effect and affecting mechanism of hypovirulent mutant strains of Bacillus subtilis,and Colletotrichum gloeosporioides to bitter rot,induced by ion implantation and magnetic field, resistance of apple variety to bitter rot,interaction between postharvest apple and C.gloeosporioides, early diagnosis technique of bitter rot and identification of apple anthracnose pathogen were studied. The results were as follows:1.Biological Characteristics on Colletotrichum gloeosporioides was studied.The results showed that the growth temperature ranged from 15℃to 35℃,with the optimum at 28℃.At 40℃,the hyphae survived for 5 days,that is,the lethal time was 5 days,while conidia still germinated after 11days.The optimum temperature for conidial germination and germ tube elongation was within 20℃to 30℃,with the optimum at 25℃for the germination and 30℃for the elongation.The maxmum biomass of Colletotrichum gloeosporioides mycelium in liquid culture was achieved at 30℃.The hyphae did not grow normally under ambient RH below 80%,while the difference of hyphal growth rate but the hyphal growth rate was not significantly different under RH over 80%.Conidia germinated only in free water or in water film,suggesting a strict germination requirement for humidity.There was a wild scope of acidity for hyphal growth and conidia germination,respectively within pH 3-11 and 2-11,with the optimum at 4.In liquid culture,the vegetative growth was not significantly different within acidity of pH 4 to 10,.During storage of apple,the developing curve of apple postharvest anthracnose displayed an unsymmetrical "S" form under a certain condition of temperature and humidity.Gompertz model was used to simulate the curve of the disease development,resulting in an extremely significant fitness.2.The molecular identification of apple anthracnose pathogens was carried out.The ITS rDNA sequences of Cg-1 and Cg-2,two isolates of apple anthracnose pathogen were determined and aligned to those of 2 C.gloeosporioides isolates on GenBank,resulting in a phylogenetic tree constructed.The results showed that Cg-1 and Cg-2 shared the highest sequence similarity(99.8%) with the known C. gloeosporioides and they grouped into the same branch with C.gloeosporioides,while the similarities to other Colletotrichum species on GenBank were only 83.1%—98.5%,confirming that the present apple anthracnose pathogen should be attributed to C.gloeosporioides.There was a 379 bp sequence residue in 3' ends of 18S rDNA of the apple anthracnose pathogen as compared with that of the known C.gloeosporioides.A specific primer for CgF1 was designed according to the 379 bp sequence and only a 1232 bp specific band could be amplified using primer pair CgF1 and ITS4.Apple fruits were inoculated with apple anthracnose pathogen.Total DNA was extracted from the diseased tissue,and an identical 1232 bp band was amplified.It was suggested that the method is applicable for detection and rapid identification of apple anthracnose pathogen.3.Affecting mechanism and effects of fungicides against postharvest anthrancnose of apple caused by C.gloeosporioides were studied.The results showed that azoxystrobin,iprodione,mancozeb, kresoxim-methyl,flusilazol,propiconazole,carbendazim,taktamer,difenoconazole and hewenqing inhibited the growth of the hyphae of C.gloeosporioides,Half effective concentration(EC50) of azoxystrobin,iprodione,mancozeb,kresoxim-methyl,nustar,propiconazole,carbendazim,and kresoxim-methyl for inhibition of growth of the hyphae was 5.12,2.32,1806.98,0.90,0.24,0.07,10.72, 0.12,and 0.49μg/mL,respectively.Mancozeb and Propiconazole exerted poor inhibition,with the conidial germination rate up to 99.3%and 98.2%,while propiconazole caused the greatest inhibition with the germination at only 74.12%.Apples were inoculated with C.gloeosporioides by puncturing on the base at 25℃indoors,sprayed with or soaked in the suspension of the conidia of the pathogen for comparison of control efficacy.The results showed that the difference between the efficacies caused by spraying and soaking were significant.As compared to other fungicides,propiconazole and kresoxim-methyl caused higher efficacy.The control efficacy of propiconazole was 100%,while that of carbendazim was much lower.The efficacy of mixed treatment of mancozeb and carbendazim at different rates on the anthrancnose was tested by pot culture method.The results showed that mancozeb+ carbendazim(9:1) resulted in the highest efficacy,with EC50 and Co-Toxicity Coefficient(CTC) for inhibiting hyphal growth at 1.768μg/mL and 692.426 respectively,and control efficacy at 45.9%.4.Some calcium salts,including CaCl2,Ca(NO3)2·4H2O and Ca(H2PO4)2,all showed inhibition effect on hyphal growth of C.gloeosporioides.The inhibition was poor when the concentration of Ca2+ was at 900μg/ml,high when the concentration was over 1000μg/ml,but the concentration lower than 600μg/ml stimulated hyphal growth.CaCl2,Ca(NO3)2·4H2O and Ca(H2PO4)2 did not inhibit conidial germination.When the concentration of Ca2+ was higher than 900μg/ml,Ca(NO3)2·4H2O,CaCl2 and Ca(H2PO4)2 resulted in inhibition rate of germ tube nelongation at 13.10%,45.75%and 46.09%,respectively,while that was lower than 600μg/ml,germ tube elongation was stimulated.Ca (H2PO4)2 and CaCl2 showed stronger inhibition than Ca(NO3)2 on germ tube elongation.5.Effect and affecting mechanism of B.subtitles mutant strains induced by ion implantation into bitter rot was studied.In a plate test,6 mutant strains obtained by ion implantation were screened out for their efficacy on the control of postharvest anthracnose of apple fruits.Under different temperatures, introduction of antagonist strain of B.subtitles by inoculation of their spores through different ways, exerted effective control of the anthrancnose on fruits.Strain BS80-6 displayed higher inhibition under room temperature than at low temperatures,with control efficacy at 33.28%after 30 day,BS100-1, BS100-6,BS80-6 and BS120-8 showed higher inhibition at lower temperatures with control efficacy at 98.36%,95.36%,95.52%and 93.52%,respectively,while BS80-1,BS100-1 and BS120-8 controlled bitter rot more effectively under changed temperatures,with efficacy at 84.59%,75.15%and 72.40%, respectively.The fermented broth showed the highest inhibition and control efficacy among the three liquid treatents.The mechanisms of the inhiibition activity of B.subtitles appeared to involve production of an antifungal substance,deformation of hypha cells and disintegration of cell walls.In addition,B.subtitles also dislpayed potentially inhibition ability of stemming the pathogen from production of some enzymes such as peroxidase(POD) and polyphenoloxidase(PPO) suggesting induceed apple resistance to C.gloeosporioides.6.Combination effects of heating plus fungicide,calcium salt plus fungicide and B.subtitles plus fungicide on control of postharvest anthracnose of apple caused by C.gloeosporioides were studied. The results showed that heating-fungicide combination resulted in the highest effucacy on bitter rot. Heating at 40℃,the effect of Propiconazole and hewenqing was the highest.Cell-free filtrate in combination with kresoxim-methyl and calcium also provided better control of anthracnose than separate application.7.Hypovirulent isolates of C.gloeosporioides were screened,with two hypovirulent isolates derived:C100-2-5,a hypovirulent isolate induced by ion implantation and C0.25-1-2,another hypovirulent isolate induced by magnetic field,and their control efficacy on apple bitter rot was studied. The control efficacy of C100-2-5 reached 55%.Enzymes activity test showed between hypovirulenct and virulent isolates were not significantly different.Primers CgF/CgR were utilized to amplify the rDNA sequences of the hypovirulent isolates.The sequencing result showed that the full-length of the rDNA sequence was 3156 bp.The mutant strain had only 1-2 nucleotide difference as compared to the original strain.They shared the sequence similarity as high as 99.8%,and the mutant strain had almost no molecular variation.RAPD and ISSR molecular markers were utilized to reveal DNA polymorphism of the original strain of apple anthracnose pathogen and its hypovirulent isolates.Thirty RAPD random primers were screened,and most primers did not display any diverse pattern except for Primer C-9. However,C-9 amplified only identical polymorphism patterns between the normal strain and its hypovirulent isolates.Almost all the 20 ISSR primers amplified multiple polymorphism patterns,but the polymorphism patterns amplified from the original strain and its hypovirulent isolates were completely identical.The molecular markers did not reveal any difference between the strains..8.The interaction between postharvest apple and C.gloeosporioides and its mechanism were studied.Resistances of five apple cultivars to bitter rot disease were identified by means of disease index.The results through Duncan's multiple range test showed that the resistence difference between the cultivars were significant(P=0.01).Hongfushi was susceptible,Gala and Qiaonajin mild susceptible, Liaofu mild resistant,while Huangjinshuai was resistant.Activities of the cell wall degrading enzymes (CWDEs) produced by C.gloeosporioides both in vitro and in vivo were studied.The results primarily demonstrated that PMG and Cx played key roles during the pathogenesis..The activities ofβ-1,3-glucanase and Chitinase in apple fruits were both induced after inoculating C.gloeosporioides,and the activity of Chitinase was increased more than those ofβ-1,3-glucanase.The activities in susceptible varieties were a little higher than those in resistant one,but the difference was not significant. The activities in susceptible cultivars kept increasing during of the whole infection process,while the activities in resistant cultivars declined gradually in late period of infection.There was no obvious change of the activities in apple fruits of CK.The postharvested fruit of three apple varieties with different resistance to apple anthracnose were inoculated with C.gloeosporioides,and tested for their activities of peroxidase(POD),polyphenoloxidase(PPO) and phenylalanine ammonia-lyase(PAL).The results showed that the activity of POD of different cultivars were all induced after inoculation,and were higher than that of CK,but that in resistant cultivars increased a lot.The maximum appeared early and maintained at high level;the activity in mild susceptible and susceptible cultivars rose slowly,the maximum appeared late,and then declined rapidly.It was also found that the activity on the infected spot(1cm) was the highest and obviously higher than that of the flesh in resistant cultivar;the activity difference in susceptible cultivars was not significant.Changes of activities of PPO and PAL showed a similar trend.In resistant variety,there were two activity peaks on the third day and on the eighth day respectively for POD,PPO or PAL.In mild susceptible and susceptible cultivars,activity peak of PAL appeared on the fourth day,that of PPO appeared on the fifth day,and that of POD appeared last.By biochemical methods,the content of total soluble sugar,organic acid,lignin and chlorogenic acid in pre-infection and post-infection apple fruit were mensurated.The results showed that before inoculation the content of total soluble sugar in healthy fruit was positively correlated to disease index of cultivar (R2=0.9978),while the contents of lignin(R2=-0.9811) and chlorogenic acid(R2=-0.9939) in healthy fruit were negatively correlated to disease index.The content of total soluble sugar and organic acid sometimes increased and sometimes decreased,the change scope was not similar in different cultivars. Meanwhile,the contents of lignin and chlorogenic acid tended to rise after inoculation,and rose more in resistant cultivar than in susceptible ones.
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