| Mango malformation disease (MMD) caused by Fusarium spp. is one of the most important mango diseases. So far, neither resistant cultivates nor MMD control methods have been found. Apparently, further researches on the genetic diversity of Fusarium mangiferae and the plant-pathogen interaction mechanisms are needed to help us in understanding the host pathogenesis and in finding possible solutions to the disease such as potential resistance genes that could be used in breeding for MMD resistance. The aim of the present study was to assess the genetic diversity of F. mangiferae using vegetative compatibility group (VCG) and inter-simple sequence repeat (ISSR); investigate the interaction mechanisms between mango and F. mangiferae though physiological and transcriptome analysis. The main results were as follows:1. Thirty-eight isolates from MM-affected mango tissues were collected from two provinces in China, on the basis of the morphology and EF-la sequences assay, all tested isolates were identified as F. mangiferae. Koch’s postulates were completed successfully with all tested F. mangiferae. Genetic diversity was assessed using VCG and ISSRs. The tested single-conidium strains were then transplanted on KClO3-containing potato sucrose agar (KPS) plates to mutagenize nit mutants and455nit mutants were recovered from the isolates by transferring chlorate-resistant sectors from KPS plates to minimal medium (MM), the VCG result showed that four mutants types were identified as nit A, nit B, nit C and nit D based on the growing situation of the mutants on three kinds of different nitrogen media, i.e., MM, nitrite medium (NM) and hypoxanthine medium (HM), among the four types, nit A was the dominant types, with the frequency of78.02%, next were nit B and nit C, with the frequency of7.91%and13.63%, respectively, and the least one was nit D, with the frequency of0.44%. The nit mutants then were paired on MM plates to test the vegetative compatibility of all isolates, thirty-eight isolates were assigned to5VCG,30isolates in VCG1,3isolates in VCG2, VCG3and VCG4contains2isolates, respectively, isolate MG33formed separate VCG5. The ISSR result showed that seventy-two bands were amplified from the38isolates by14ISSR primers, of which52bands (72%) were polymorphic. The size of the amplified products ranged from300to2000bp. The genetic similarities between isolates ranged from0.5972to0.9862, among those isolates MG16 and MG17were the most similar (0.9862), whereas isolates MG07and MG33were the least similar (0.5972). At a genetic similarity of0.76, the unweighted pair-group method with arithmetic mean analysis separated the isolates into five distinct clusters,33isolates in cluster V, isolates MG25and MG35in cluster IV, isolates MG36, MG07, MG33formed separate clusters Ⅲ, Ⅱ, Ⅰ, respectively. It indicated that there were high genetic diversity among the isolates of F. mangiferae in China and relationships in isolates of F. mangiferae collected from different mango cultivars and locations are not obviously related based on the VCG and ISSR data.2. The biological characteristics of F. mangiferae were studied. The results showed that there were discrepancies in pathogenicity among all tested isolates. The best media on mycelial growth rate, amount of mycelial growth and sporulation quantity of F. mangiferae were OMA, liquid PDA and PSA, respectively. Optimum temperature range on mycelial growth and spore germination was25℃-28℃, influence of light and pH4-10on mycelial growth, sporulation and spore germination of F. mangiferae were not obvious. Sucrose was the best carbon source and yeast extraction was the best nitrogen source to the growth of F. mangiferae. The lethal temperture of mycelia and spore of F. mangiferae was53℃and57℃, respectively. The result of toxicity test showed that25%Prochloraz EG had the best control effect on mycelial growth, its EC50was1.2004μg·mL-1and EC95was2.6239μg·mL-1and the best fungicide on spore germination was25%Azoxystrobin SC, its EC50was0.5312μg·mL-1and EC95was2.9741μg·mL-1.3.The result of an investigation on interaction of physiological mechanisms between mango and F. mangiferae showed that:①The contents of photosynthetic pigments, total soluble sugar, reduced sugar, sucrose, glucose incessantly rose in the earlier period then declined and the content of starch and cellulose was decreased after inoculation; activities of neutral invertase (NT) and SUS (sucrose synthase)-synthetic were increased at primary period of inculation, activities of NI, SUS-synthetic and sucrose phosphate synthase were decreased and SUS-cleavage rose at late period after inculation.②The content of mineral elements were changed after infection of F. mangiferae, in which contents of eight elements were continuously increased, of which N, K, Fe and Mn were increased greater than P, Mg, B and Zn; the contents of Cu and S had no obvious change and the content of Ca was continuously declined after inoculation.③The content of soluble protein and RNA increased significantly at the earlier period, rose to a peak of45d, then declined and the content of DNA rose after inoculation.④Infection of F. mangiferae promoted O2production rate and increased H2O2and malondialdehyde (MDA) contents. The content of glutathione (GSH) declined quickly; the activities of peroxidase (POD), catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR) increased at the earlier period and kept at high level at late period, and the activity of ascorbate peroxidase (APX) increased at the early period then increased rapidly after inoculation.⑤The contents of endogenous IAA and GA3rose rapidly and were significantly higher than controlat at whole period after inoculation, ABA content was rose at the late period after inoculation.⑥Total contents of phenolics and flavonoids and activity of phenylalanine ammonia-lyase (PAL) were significantly increased in45days after inoculation, then decreased quickly, activity of polyphenol oxidase (PPO) did not change obviously at beginning after inoculation, then increased continuously.4. Using the Illumina HiSeqTM2000platform, the transcriptomes of healthy and infected mango bud were sequenced. Through Blastx comparation to nr、Swiss-Prot、 KEGG and COG databases, the functional annotation of the differentially expressed genes (DEGs) were obtained. DEGs were then carried out into GO functional analysis and KEGG pathway analysis. The results showed that:①a total of119,815unigene were received from the two samples, with the average length was880bp and N50value1546. Through the transcriptomes analysis with the RPKM (reads per kb per million reads method),29,878DEGs were acquired. Taking corrected-pvalue<0.05as a threshold, DEGs involved in22enrichment pathway, most of which were closely related with the plant’s response to stress.②A total of153DEGs involved in starch and sucrose metabolism, in which most of those DEGs coded racemase and epimerase, hydrolase and intramolecular transferase, taken part in the glucose catabolic process, carbohydrate metabolic process, pyruvate metabolic process, nucleotide metabolic process and so on.③In the antioxidant activity process, twenty-four DEGs which coded enzymes invlved reactive oxygen metabolism were acquired with ratio larger than five and nineteen DEGs were up-regulated. It demonstrated that reactive oxygen metabolism played an important role in the mango-F. mangiferae interaction.④With ratio larger than ten, fifty-three DEGs related to phenolic metabolism were acquired, forty of which were up-regulated. It indicated that mango may protect itself from F. mangiferae by accelerating the synthesis of secondary substances, such as lignin and phenolic compounds.⑤Calcium signal transduction pathways, salicylic acid (SA) pathway and mitogen-activated protein kinase (MAPK) signal pathways were down-regulated in the mango-F. mangiferae interaction. RPM1, a important gene related to plant disease resistance, was down-regulated, which was probably main reason to the susceptibility of mango to F. mangiferae. |
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