| Gray mold,caused by Ascomycotina fungus Botrytis cinerea,is an important plant disease worldwide.This pathogen has a widely distributed host origin infecting more than 200 hosts in agriculture,including ornamental flowers,fruits and vegetables,and resulting in severe economic loss.Currently,because no resistance crop varieties are available,and the pathogen had high risk characteristics,such as high capacity of sporulation and easily genetic variation,managing this disease is relied on botryticides.SYP-1620 and fluopyram are two novel respiratory inhibitors.SYP-1620,developed by Shenyang Research Institute of Chemical Industry and registerted in 2009 in China,is a novel broad-spectrum systemic fungicide by inhibiting complex Ⅲ(Qo)of fungal mitochondrial respiration chain.However,biological characteristics of SYP-1620 against B.cinerea have not been studied.Fluopyram,developed by Bayer Investment(China)Limited Cooperation and registerted in 2012 in China,is a novel succinate dehydrogenase(complex Ⅱ of eclectron transportation chain)inhibitor(SDHI)via inhibiting fungal mitochondrial respiratory chain.It is used to control more than 70 diseases caused by Botrytis spp.,Sphaerotheca fuliginea,Sclerotinia spp.and Monilia spp..However,fluopyram is not well documented in controlling gray mold.Therefore,biology of B.cinerea against SYP-1620 and fluopyram was studied in this paper to reveal the difference and similarity among the two inhibitors and the same corresponding group fungicides,and develop scientific use techniques to manage the populations of B.cinerea.Main results were presented as follows.Firstly,baseline sensitivity of B.cinerea to SYP-1620 and its potential resistance risk assessment were studied.The results showed that SYP-1620 has high inhibition activity on mycelial growth and spore germination in vitro,the baseline sensitivity of 108 B.cinerea isolates to SYP-1620 was set up with a mean EC50 value of 0.0130±0.0109 μg/mL for mycelial growth,0.01147±0.0062 μg/mL for spore germination,respectively.Greenhouse experiment of SYP-1620 showed that both protective and curative activity was excellent,but protective efficacy was superior to curative efficacy on the leaves of strawberry and cucumber in vivo.The fitness of SYP-1620-resistant mutant was inferior to sensitive isolates.SYP-1620 had a positive cross-resistant with azoxystrobin with the same mode of action,but had no cross-resistant with other different mode of action fungicides.Sequencing analysis suggested that a single point mutation(G143A)in the cytochrome b gene always occurred in SYP-1620 resistant isolates.In short,the results suggested that B.cinerea has high resistance risk to SYP-1620,and SYP-1620 had similar toxicology with the other QoIs funficides.Secondly,the resistance risk of B.cinerea to fluopyram and its resistance detection techniques were involved.The results showed that baseline sensitivity of fluopyram against 100 single-spore B.cinerea isolates was a unimodal curve with a mean of EC50 value of 1.94±1.55 μg/mL for mycelial growth.The fitness of fluopyram-resistant isolates in field was significantly inferior to sensitive isolates.Sequencing analysis showed that the mutation occurred in SdhB subunit leading to an amino acid alteration of arginine to histidine(H272R)in resistance isolates.But fluopyram has no cross-resistance with the other SDHIs boscalid,and other fungicide with different mode of action.Thus,these results suggested that resistance risk of B.cinerea to fluopyram was at the low to medium level.Furthermore,an allele specific oligonucleotides-PCR(ASO-PCR)with mismatches at the 3’ terminal of anti-sense primer was developed to detect fluopyram-resistance genotype B-H272R from field.The technique is a rapid,effective method for detecting the specific resistance genotype in field.Finally,the relationship between the amino acid polymorphism of SdhC subunit from B.cinerea and SDHIs were studied.Our previous studies showed that significant difference in sensitivity against SDHIs in B.cinerea isolates,which have never exposed to the SDHIs,was observed.We inferred that the sensitivity difference maybe results from diversity of SDH,a target of SDHIs.Thus,the whole sequences of SdhB,SdhC and SdhD subunits of 52 B.cinerea isolates collected from different geographical districts in China were analyzed.Compared with reference B05.10,amino acid polymorphism occurred only in the SdhC subunit and the 52 isolates were categorized into the five types,from CO to C4.The five types of wide-type isolates had a significant difference in sensitivity against SDHIs and other fitness parameters.To eliminate the different genetic background,five types of mutants were obtained(R-CO to R-C4)by gene replacement technique,and validated by PCR and Southern Blotting.The results showed that EC50 values of R-CO mutants were significantly higher than the other 4 types of mutants.The five types of mutants had a significant difference in mycelial growth rate,sporulation,utilization of the carbon source,and virulence on the leaves of tomato and strawberry,but no significant difference in the virulence on fruits of apple and onion.Additionally,tolerance of oxidative stress induced by 10 μg/mL paraquat in R-CO mutants decreased significantly less than the other 4 types of mutants.In conclusion,SYP-1620 and fluopyram have powerful inhibition against B.cinerea,it is recommended to control gray mold in field,but they should be alternatively used or mixtured with different mode of action fungicides to delay commercial longevity of the two fungicides.The data supported that SYP-1620 had the same mode of action with the other strobilurin fungicides,however,no cross resistance existed between fluopyram and boscalid,this may result from difference mechanism between the succinate dehydrogenase inhibitors.Genetic diversity of SdhC subunit is involved in the intrinsic sensitivity against SDHIs. |
