Development of real-time PCR method for detection of Xanthomonas campestris pv. juglandis causing walnut bacterial blight

Walnut is one of the important economic tree species in the world. For the past few years, with the continuous expansion of walnut planting area, walnut bacterial blight caused by Xanthomonas campestris pv. juglandis has critically affected walnut production and caused economic losses. In this research, by comparing the gene sequences of X. campestris pv. juglandis and its closely related bacterial species, the 16S rDNA gene sequence was selected, and the specific primers were evaluated using PCR. Subsequently, the real-time fluorescence PCR specific detection method of X. campestris pv. juglandis was established and optimized. The specificity of real-time PCR was verified for 23 strains isolated from walnut and its surrounding soil, including eight target pathogens. The specific amplification was possible even when the cDNA template concentration was 7.0×10-5 µg/mL, hence, the detection sensitivity of the standard method is 7.0×10-5 µg/mL. The reliability of the established real-time PCR detection method in field detection was verified by the identification of target bacteria in 8 randomly collected healthy leaves and leaves of suspected walnut blight. This method laid a foundation for the early diagnosis of X. campestris pv. juglandis

Pathogenesis mechanism of Pestalotiopsis funerea toxin (Pf-toxin) on the plasmalemma of needle cells of different pine species

The Pf-toxin (C5H11O5N) has been genetically associated with the pathogenesis mechanism in plasmalemma cells of pine needles in previous reports. In this study, a toxin was obtained from Pestalotiopsis funerea (called Pf- toxin) by concentrating and column chromatography. Responses of the needles of eight pine species against the toxin were investigated. The O2- production rate, malondialdehyde (MDA) content, fatty acid composition, relative conductivity, and lesion length of the needles were determined. The severest damage and lipid peroxidation were exhibited by the needle plasmalemma of Pinus massoniana, Pinus yunnanensis, and Pinus tabuliformis. Pinus elliottii and Pinus taeda followed. Pinus armandi, Pinus radiata and Pinus thunbergii came last. The resistance capability of resistant species against the Pf-toxin precedes that of susceptible species. Keywords: Pestalotiopsis funerea, Pestalotia needle blight, Pinus, resistance. African Journal of Biotechnology Vol. 11(29), pp. 7397-7407, 10 April, 201

Cloning and Expression of the Chitinase Encoded by <i>ChiKJ406136</i> from <i>Streptomyces Sampsonii</i> (Millard &amp; Burr) Waksman KJ40 and Its Antifungal Effect

The present study demonstrated that the chitinase gene ChiKJ406136 of Streptomyces sampsonii (Millard &amp; Burr) Waksman KJ40 could be cloned using a PCR protocol and expressed in Escherichia coli (Migula) Castellani &amp; Chalmers BL21 (DE3), and the recombinant protein had antifungal effect on four forest pathogens (Cylindrocladium scoparium Morgan, Cryphonectria parasitica (Murrill) Barr, Neofusicoccum parvum Crous, and Fusarium oxysporum Schl.) and also had the biological control effects on Eucalyptus robusta Smith leaf blight, Castanea mollissima BL. blight, Juglans regia L. blight and J. regia root rot. The results showed that ChiKJ406136 was efficiently expressed and a 48 kilodalton (kDa) recombinant protein was obtained. No significant change in protein production was observed in the presence of different concentrations of IPTG (isopropyl-b-D-thio-galactoside). The purified protein yield was greatest in the 150 mmol/L imidazole elution fraction, and the chitinase activities of the crude protein and purified protein solutions were 0.045 and 0.033 U/mL, respectively. The antifungal effects indicated that mycelial cells of the four fungi were disrupted, and the control effects of the chitinase on four forest diseases showed significant differences among the undiluted 10- and 20-fold dilutions and the control. The undiluted solution exhibited best effect. The results of this study provide a foundation for the use of S. sampsonii as a biocontrol agent and provides a new source for the chitinase gene, providing a theoretical basis for its application

Tandem Mass Tags Quantitative Proteome Identification and Function Analysis of ABC Transporters in <i>Neofusicoccum parvum</i>

Neofusicoccum parvum can cause twig blight of the walnut (Juglans spp.), resulting in great economic losses and ecological damage. We performed proteomic tandem mass tags (TMT) quantification of two Neofusicoccum parvum strains with different substrates, BH01 in walnut substrate (SW) and sterile water (SK), and BH03 in walnut substrate (WW) and sterile water (WK), in order to identify differentially expressed proteins. We identified 998, 95, and 489 differentially expressed proteins (DEPs) between the SK vs. WK, SW vs. SK, and WW vs. WK comparison groups, respectively. A phylogenetic analysis was performed to classify the ABC transporter proteins annotated in the TMT protein quantification into eight groups. Physicochemical and structural analyses of the 24 ATP-binding cassette (ABC) transporter proteins revealed that 14 of them had transmembrane structures. To elucidate the functions of these transmembrane proteins, we determined the relative expression levels of ABC transporter genes in strains cultured in sodium chloride, hydrogen peroxide, copper sulfate, and carbendazim mediums, in comparison with pure medium; analysis revealed differential upregulation. To verify the expression results, we knocked out the NpABC2 gene and compared the wild-type and knockout mutant strains. The knockout mutant strains exhibited a higher sensitivity to antifungal drugs. Furthermore, the virulence of the knockout mutant strains was significantly lower than the wild-type strains, thus implying that NpABC2 plays a role in the drug resistance of N. parvum and affects its virulence