| Peach brown rot and peach anthracnose,caused by Monilinia spp.and Colletotrichum spp.,respectively,can be severe problems in many areas of peach production in the world.In this study,these two main pathogens of peach were used as research materials to explore the resistance status to several major single-site fungicides including DMI fungicides.M.fructicola is the dominant species causing peach brown rot and widely distributed in China.The sensitivity of M.fructicola isolates from important peach-growing provinces in China to four main site-specific fungicides as mentioned above,was determined.The mechanism of DMI resistance in M.fructicola was investigated.A Loop-mediated Isothermal Amplification(LAMP)method was then developed.The sensitivity of all Colletotrichum spp.known to cause anthrcacnose of peach in southeastern USA to DMI fungicides was also determined.The mechanism of DMI inherent resistant species Colletotrichum truncatum was also investigated.The study provided a scientific basis and technical support for management of peach brown rot and peach anthracnose.The main results were as followed:(1)Monilinia fructicola(G.Wint.)Honey is the dominant species in China,and can cause brown rot of peach in many production areas of the world.Fungicides are increasingly utilized to minimize such losses.In current study,eighty isolates of M.fructicola were collected from commercial peach orchards located in five provinces in China and the sensitivity to carbendazim,azoxystrobin,tebuconazole,and boscalid was determined.Resistance to carbendazim was detected only in the Yunnan province in 15 of 16 isolates.Characterization of carbendazim-resistant isolates revealed stable resistance,no fitness penalty,and negative cross resistance to diethofencarb.Resistant isolates produced disease symptoms on detached fruit sprayed with label rate of formulated carbendazim and possessed the amino acid mutation E198 A in ?-tubulin(TUB2).Resistance to azoxystrobin was detected in 3 of 10 isolates from Fujian.In contrast to carbendazim resistance,azoxystrobin resistance was unstable,associated with a fitness penalty,and not associated with mutations in the target gene Cytochrome b(Cyt b).The concentration at which mycelial growth is inhibited 50%(EC50)values of the azoxystrobin-sensitive isolates were 0.02 to 1.94 ?g/mL,with a mean value of 0.54 ?g/m L.All isolates were sensitive to tebuconazole,with a mean EC50 value of 0.03 ?g/m L.The EC50 values for boscalid were 0.01 to 3.85 ?g/m L,with a mean value of 1.02 ?g/mL.Our results indicate that MBCs,QoIs,DMIs,and SDHIs are likely to be very effective in controlling brown rot in many peach production areas in China,but the resistance to MBCs is emerging in some areas.(2)The genetic element ‘Mona' has been shown previously to be associated with resistance to DMIs in M.fructicola.In this study,the promoter activity of the Mona element was demonstrated genetically and the core activity region was narrowed down to a 20-bp through a series of deletions.(3)A Loop-mediated Isothermal Amplification(LAMP)method was developed for rapid detection of Mona as well as the resistance to DMIs.The time required to complete the assay was about 75 min and the result could be visualized by adding SYBR Green I.The method was highly specific to detect the Mona in the upstream of MfCYP51,thus could successfully distinguish the resistant isolates from sensitive ones in the total of 51 M.fructicola samples.The assay did not amplify DNA from other six fungal pathogens of peach.The assay had a detection limit of 10 fg of purified DNA,which is 100 fold more sensitive than conventional PCR method.The LAMP method also distinguished resistant isolates from sensitive ones when fungal mycelia and conidia were directly used as template.Considering its reliable results,easy operation and lower cost,the LAMP assay could be used for in-field diagnosis of DMI fungicide resistance.(4)Few fungicides are effective against anthracnose disease caused by Colletotrichum species and emerging resistance makes the search for chemical alternatives more relevant.Isolates of the Colletotrichum acutatum species complex were collected from South Carolina and Georgia peach orchards and phylogenetic analysis of the combined Internal Transcribed Spacer region,glyceraldehyde-3-phosphate dehydrogenase,and beta-tubulin gene sequences separated the isolates into Colletotrichum nymphaeae and Colletotrichum fioriniae.The sensitivity of these and three other Colletotrichum species from peach,including Colletotrichum fructicola,Colletotrichum siamense,and Colletotrichum truncatum to DMI fungicides difenconazole,propiconazole,tebuconazole,metconazole,flutriafol,and fenbuconazole was determined.C.truncatum was resistant to tebuconazole,metconazole,flutriafol,fenbuconazole,and C.nymphaeae was resistant to flutriafol and fenbuconazole with the EC50 values more than 100 ?g/mL.C.fructicola and C.siamense were sensitive to all DMI fungicides(EC50 values ranging from 0.2 to 13.1 ?g/m L),and C.fioriniae-subgroup 2 was less sensitive to DMI fungicides(EC50 values ranging from 0.5 to 16.2 ?g/mL)compared to C.fioriniae-subgroup 1(EC50 values ranging from 0.03 to 2.1 ?g/mL).Difenconazole and propiconazole provided the best control efficacy in vitro to all five species with EC50 values ranging from 0.2 to 2.7 ?g/mL.Tebuconazole and metconazole were effective against all Colletotrichum species,except for C.truncatum.The highly efficient activity in vitro of some DMI fungicides against Colletotrichum species may be exploited for improved anthracnose disease management of peach.(5)Sequences of CYP51 A and CYP51 B genes were cloned from DMIs inherent resistant C.truncatum isolates for the first time.Amino acid substitutions of H373 N,M376L,S511 T from CYP51 B,and L208 Y,H238R,S302 A,I366L from CYP51 A are suspected to be involved in DMIs resistance. |
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