A Specific High Toxicity of Xinjunan (Dioctyldiethylenetriamine) to Xanthomonas by Affecting the Iron Metabolism.

Xanthomonas spp. encompass a wide range of phytopathogens that brings great economic losses to various crops. Rational use of pesticides is one of the effective means to control the diseases. Xinjunan (Dioctyldiethylenetriamine) is structurally unrelated to traditional bactericides, and is used to control fungal, bacterial, and viral diseases with their unknown mode of actions. Here, we found that Xinjunan had a specific high toxicity toward Xanthomonas spp., especially to the Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial leaf blight. Transmission electron microscope (TEM) confirmed its bactericidal effect by morphological changes, including cytoplasmic vacuolation and cell wall degradation. DNA synthesis was significantly inhibited, and the inhibitory effect enhanced with the increase of the chemical concentration. However, the synthesis of protein and EPS was not affected. RNA-seq revealed differentially expressed genes (DEGs) particularly enriched in iron uptake, which was subsequently confirmed by siderophore detection, intracellular Fe content and iron-uptake related genes transcriptional level. The laser confocal scanning microscopy and growth curve monitoring of the cell viability in response to different Fe condition proved that the Xinjunan activity relied on the addition of iron. Taken together, we speculated that Xinjunan exerted bactericidal effect by affecting cellular iron metabolism as a novel mode of action. IMPORTANCE Sustainable chemical control for rice bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae need to be developed due to limited bactericides with high efficiency, low cost, and low toxicity in China. This present study verified a broad-spectrum fungicide named Xinjunan possessing a specific high toxicity to Xanthomonas pathogens, which were further confirmed by affecting the cellular iron metabolism of Xoo as a novel mode of action. These findings will contribute to the application of the compound in the field control of Xanthomonas spp.-caused diseases, and be directive for future development of novel specific drugs for the control of severe bacterial diseases based on this novel mode of action.

Vegetative Compatibility of Fusarium graminearum Isolates and Genetic Study on Their Carbendazim-Resistance Recombination in China.

ABSTRACT Monoconidial isolates of 33 carbendazim-sensitive isolates and 31 carbendazim-resistant isolates of Fusarium graminearum were selected from three regions of China for vegetative compatibility group (VCG) analysis. A total of 213 and 224 nit mutants were recovered from the 33 sensitive and the 31 resistant isolates, respectively. Of all the nit mutants, the frequency of the different phenotypes was 44.6, 46.5, 5.7, and 3.2% for nit1, nit3, nitM, and nitA, respectively. VCG analysis identified 30 different VCGs among the 33 sensitive- and the 31 carbendazim-resistant isolates, with VCG diversity 0.91 and 0.97, respectively. Both, a carbendazim-sensitive and a -resistant isolate from the same field belonged to the same VCG. In all then, a total of 59 VCGs were identified among the 64 isolates with an overall VCG diversity 0.92. Direct hyphal fusion was observed in six pairs of vegetatively compatible complements, which is evidence of heterokaryon formation. It was hypothesized that carbendazim resistance could not be transferred by hyphal fusion or there is a small chance to be transferred between two compatible isolates. Three stable sexual recombinants of F. graminearum were randomly chosen from each of the three genetic crosses to study their biological properties. There were no significant differences in mycelial linear growth and pathogenicity between recombinants and their parents, but they differ in sporulation ability and capacity to produce perithecia. We concluded that sexual recombination presumably played a role in the development of carbendazim resistance under field conditions.

[Induction and genetic analysis of laboratory mutants of Gibberella zeae resistance to carbendazim].

The mutants of wild-type Gibberella zeae resistance to carbendazim were generated in laboratory by ultra-violet (UV) irradiating and fungicide taming. Two levels of resistance (low, LR; high, HR) were identified among these mutants. LR mutants could grow at the critical concentration of 1.4 +/- microg/mL of carbendazim while completely inhibited above 10 microg/ mL concentration, but this phenotype of isolates was not detected in the field. HR mutants could grow at 100 microg/mL and showed negative cross-resistance with N-phenylcarbamate fungicides, such as N-( 3,5-dichlorophenyl) carbamate (MDPC) whereas the LR mutants were not. Moderate resistant (MR) mutants which could grow fast at 10 microg/mL, but completely inhibited at 100 microg/mL, were not generated in laboratory. HR mutants of field MR isolate were also generated by fungicide taming on 100 microg/mL of carbendazim, but these mutants did not express negative cross-resistance to MDPC. The genetic study suggested that the carbendazim-resistance in these mutants could be steadily inherited by asexual and selfed reproduction, and the resistance was controlled by the same single major gene both in laboratory mutants and field resistant isolates, the different levels of resistance or the same resistant level in different strains maybe conferred by mutations at different sites or one site with different allelic mutations. The gene compensates for the sensitivity to MDPC was allelic to that governs the carbendazim resistance, but the mutation for increasing sensitivity to MDPC in this gene could get the pathogen highly resistant to carbendazim.

[Genetics of resistance to carbendazim in Gibberella zeae].

According to the ability of the field isolates of Gibberella zeae to grow on the PSA with varying carbendazim(MBC) concentrations, three sensitivity levels of isolates were determined in vitro. The sensitive isolates(S) could grow at 0.5 microgram/ml, but were completely inhibited at 1.4 micrograms/ml. The moderate resistant isolates (MR) could grow fast at 1.4 micrograms/ml and slow at 50 micrograms/ml, but could not grow at 100 micrograms/ml. The high resistant isolates(HR) could grow faster than R at 50 micrograms/ml, and also could grow at 100 micrograms/ml. No low resistant isolates, that could grow fast at 1.4 micrograms/ml but could not grow at 50 micrograms/ml, were found among the field isolates. The genetics of resistance to carbendazim in G. zeae was investigated by analyzing the sensitivity of sexual outcrossed progeny to MBC. The nitrate non-utilizing mutant (nit) as an another added genetic marker was used to select the out-crossed perithecium from self-crossed perithecia. Seven crosses were tested in all, including cross between S x S, MR x S, MR x MR, HR x S, as well as HR x MR. The results showed that no recombinant phenotype was found among all progeny of seven crosses. Progeny segregation of crosses between the parents with different sensitive levels(i.e. MR x S, HR x S, HR x MR) clearly fit a 1:1 ratio of two parental phenotypes, and no segregation was found in the crosses of S x S and MR x MR. So it can be concluded that the two levels of MBC resistance in G. zeae are conferred by two loci mutations or one locus with different allelic mutations that constitute a polymorphic series in a single Mendelian gene. In these isolates, the MBC resistance is not affected by modifying genes or cytoplasmic components.