Induction Of Resistance To Colletotrichum Gloeosporioides And Its Resistance Mechanis

Anthracnose,caused by Colletotrichum gloeosporioides,is one of the most important fungal diseases of mango.Pseudolaric acid B（PAB）,a natural diterpenoid acid isolated from traditional chinese herb Cortex Pseudolaricis with novel structure,showed the many kinds of pharmacological action and strong fungicidal activity against mycelial growth of plant pathogenic fungi with a broad spectrum.In our previous study,PAB was more effective to inhibit mycelial growth and spore germination for C.gloeosporioides in vitro than benzimidazole fungicides carbendazim,with EC₅₀ of0.882μg/m L and 3.8μg/m L.PAB above is also effective on carbendazim-resistant C.gloeosporioides.Further study showed that PAB could affect the mitotic process of C.gloeosporioides and reduce the number of mitosis during spore germination,as with the carbendazim..Thus we suspected that the target for drugs of PAB and tubulin inhibitors carbendazim were simliar.But its action sites had different.Therefore this paper selects the PAB for the study.The PAB-resistant C.gloeosporioides mutants were obtained by drug domestication.Then,the fitness studies and stability of the PAB-resistant C.gloeosporioides mutant were studied,and assessed the cross resistance of PAB.In addition,the mechanisms antimicrobial action of C.gloeosporioides to PAB were study from resistance by whole genome resequencing and molecular docking and molecular biology techniques.The mutation sites were found in PAB-resistant mutants.These results provide clues to understand the mechanisms antimicrobial action of C.gloeosporioides to PAB.The results are as follows:（1）The 18 PAB-resistant C.gloeosporioides mutants with stable inheritance were obtained by drug domestication.The frequency of resistant was 1.69×10^-7.The resistance level classification of mutants had found that the 8 mutants were divided into low resistance levels,with EC₅₀ value range from 4.48μg/mL～9.06μg/mL and the resistance multiples ranged from 4.88～9.87.The EC₅₀ values of PAB on mycelia growth of moderately resistant isolates QHZ,B1Z,Q2C1Z,X1Z,A1Z,E3Z,HZ,F1Z were 48.24,21.04,17.00,2.91,25.19,32.35,25.17 and 25.46μg/m L,respectively,and the resistance multiples ranged from 14.06～52.52.In addition,the high-PAB resistant mutants M5 and M9 were obtained,with EC₅₀>100μg/m L.（2）Confirmed with the different fitness of parent isolate and resistant mutants.The result showed that a total of resistant and parental strain could normal growth on PDA at 15-35℃,and the faster for mycelia growth was 25℃.However,the tests found that the mycelia linear growth rates were different of mutants with the different resistant level.The law is low antibacterial strains>parental strains>medium antibacterial strains>high resistant strains.The environmental stress experiment result suggest that the parent strain was more sensitive to 0.02%SDS,and its inhibition rate was as high as 70%.But under metal ion stress（Na Cl,KCl and Ca Cl₂）,the resistant mutant strains were more sensitive.In addition,there was a positive cross-resistance between PAB and carbendazim in C.gloeosporioides,but there was no cross resistance relationship existed between PAB and prochloraz,tebuconazole,triadimefon or pyraclostrobin..（3）We found that the tubulin may be a target for PAB.Based on resequencing data,we found a nucleotide mutation at position 1289 ofα1-tubulin gene of PAB-resistant mutants M5 and M9 of C.gloeosporioides,which was the mutation of 430the amino acid of from Leu（L）to Pro（P）.Then,we based on tubulin crystal structure（PDB code:5NM5）,α-tubulin AQ,α-tubulin A-M9 andβ1-tubulin Q complex structures were constructed,and PAB was docked into the cavity ofα-tubulin AQ,α-tubulin A-M9 andβ1-tubulin Q.The results showed that PAB bound to the colchicine site of tubulin,and l430p decreased the affinity between tubulin and PAB.This study confirmed that the resistant strain and the parent strain had similar biological fitness,and found that PAB had positive cross resistance to tubulin inhibitor carbendazim.Subsequent genome resequencing and molecular docking showed that tubulin may be an action target of PAB,but its specific action site needs to be further understood.