Trichoderma harzianum produces nonanoic acid, an inhibitor of spore germination and mycelial growth of two cacao pathogens

An isolate of Trichoderma harzianum Rifai from an infected cacao pod produces and secretes nonanoic (pelargonic) acid into a liquid culture medium. Nonanoic acid (NA) was very inhibitory to spore germination and mycelial growth of two cacao pathogens, Crinipellis perniciosa Stahel and Moniliophthora roreri Cif. H.C. Evans. It was highly active causing 75% inhibition of spore germination in an in vitro assay at a rate as low as 0.09 μM for M. roreri and 0.92 μM for C. perniciosa. Mycelial growth was comparatively less sensitive to inhibition, but still there was a 75% reduction in growth with 0.62 μM in M. roreri and 151 μM NA in C. perniciosa. In contrast, NA did not affect Trichoderma mycelial growth or spore germination at concentrations that were inhibitory to the pathogens. 6-pentyl-α-pyrone was also produced and secreted into the medium by T. harzianum, however; it was not antagonistic to the cacao pathogens. Although a number of metabolites produced by Trichoderma spp. have been identified in the past, this is the first report of NA production and secretion by any Trichoderma. The results suggest that NA may play a role in the successful use of some Trichoderma spp. isolates in the biocontrol of fungal diseases of plants.     

Transgenic cucumber expressing an endogenous class III chitinase gene has reduced symptoms from Botrytis cinerea

A class III chitinase gene (CHI2) is induced in cucumber plants (Cucumis sativa L.) in response to infection by pathogenic microorganisms. Infection of Botrytis cinerea, causal agent of gray mold disease on cucumber, also induces CHI2 expression. To investigate whether CHI2 is involved in resistance to gray mold disease, transgenic cucumber plants were produced to overexpress the CHI2 gene. One line was analyzed in detail in terms of disease resistance. The transgenic cucumber plant (CC2) constitutively expressed CHI2 and reduced the symptoms of B. cinerea for 4 days after inoculation compared with nontransgenic plants. However, this inhibitory effect was not absolute, and CC2 eventually developed serious disease symptoms. Chitinase activity of the crude extract from CC2 leaves was higher than that from nontransgenic plants. A high-molecular-weight fraction containing CHI2 from CC2 leaves had fungistatic activity against B. cinerea. Interestingly, the low-molecular-weight fraction from CC2 leaves with CHI2 removed also had fungistatic activity against B. cinerea. Not only the introduced chitinase activity but also the endogenous defense reactions activated by overexpression of CHI2 may be involved in the enhanced gray mold disease resistance in CC2.     

Synergism between potassium sorbate dips and brief exposure to high CO2 or O2 at curing temperature for the control of citrus postharvest green and blue molds

Synergistic effects and very effective control of citrus postharvest green and blue molds, caused by Penicillium digitatum and Penicillium italicum, respectively, were observed on artificially inoculated ‘Valencia’ oranges and ‘Clemenules’ and ‘Ortanique’ mandarins after a potassium sorbate (PS) treatment was followed by 2 days of storage in atmospheres of elevated CO₂ or O₂ at a curing temperature. A combined treatment consisting of 60-s dips in aqueous solutions of 3% PS heated to 62 °C was followed by 48-h exposure to air, 15 kPa CO₂ or 30 kPa O₂ at 33 °C. Control treatment was a 60-s water dip at 20 °C followed by 24-h exposure to air at 20 °C. Synergism was observed on citrus fruit either incubated at 20 °C for up to 22 days, simulating direct commercialization, or stored at 5 °C for up to 45 days, simulating commercial cold storage. This research offers potential new tools to the citrus industry for implementation of nonpolluting integrated postharvest disease management programs, especially devoted to high added value organic markets or export markets with zero residue tolerance.     

九里香层析物的抑菌活性及成分分析

以石油醚为洗脱液，对九里香氯仿萃取物进行硅胶柱层析分离，得到27个组分。采用生长速率法测定九里香层析物对5种供试病原菌的抑制作用。结果表明，在质量浓度为0.5 g/L时，组分26对5种供试病原菌有较好的抑制活性，对香蕉炭疽菌的抑制率达到89.46%，对香蕉炭疽菌的EC50为0.078 6 g/L。GC-MS分析表明，组分26的主要成分是7-甲氧基-8-异戊烯基香豆素，占92.00%。     

Analysis of the gacS-gacA regulatory genes of spontaneous mutants of Pseudomonas fluorescens biocontrol strain PfG32R

Pseudomonas fluorescens strain PfG32R, a potential biocontrol agent against soilborne pathogens, frequently loses its antifungal activity and ability to produce enzymes. To characterize genetically the instability of these bacterial functions, we analyzed gacS and gacA genes of PfG32R and three spontaneous mutants of PfG32R (NR1, NR9, and ASW6), which had lost their ability to produce proteases and antifungal activities. The gacS and gacA sequences of PfG32R had 77%–89% and 78%–87% homology, respectively, with several known gacS and gacA homologues. All three spontaneous mutants were subjected to complementation analysis. Introduction of clones containing an intact gacS of PfG32R and another P. fluorescens strain Pf-5 as well as strain tea632 of P. syringae pv. theae complemented all three mutants restoring protease and antifungal activities, indicating a mutation in gacS. In sequencing analysis, the mutants had a deletion or change in amino acids in the conserved sensor kinase domains of GacS. The three mutants maintained both their antibacterial activity against Ralstonia solanacearum and Clavibacter michiganensis ssp. michiganensis and siderophore production, indicating that they are not controlled by the GacS/GacA system. The sequences reported in this article have been deposited in the DDBJ database under accession numbers AB219364 and AB219365.     

Concentrations of constitutive alk(en)ylresorcinols in peel of commercial mango varieties and resistance to postharvest anthracnose

Mature green mango fruits of commercially important varieties were screened to investigate the levels of constitutive antifungal compounds in peel and to assess anthracnose disease after inoculation with Colletotrichum gloeosporioides. High-pressure liquid chromatography was used to quantify the levels of 5-n-heptadecenylresorcinol and 5-n-pentadecylresorcinol in the peel extracts. The fruit peel of the varieties ‘Kensington Pride’ and ‘Keitt’ were observed to have the highest levels of both 5-n-heptadecenylresorcinol (107.3–123.7 and 49.9–61.4 μg/g FW, respectively) and 5-n-pentadecylresorcinol (6.32–7.99 and 3.30–6.05 μg/g FW, respectively), and the fruits of the two varieties were found to have some resistance to postharvest anthracnose. The varieties ‘Kent’, ‘R2E2’, ‘Nam Doc Mai’, ‘Calypso’ and ‘Honey Gold’ contained much lower concentrations of resorcinols in their peel and three of these varieties were found to be more susceptible to anthracnose. Concentrations of 5-n-heptadecenylresorcinol were significantly lower at the ‘sprung’ and ‘eating ripe’ stages of ripening compared to levels at harvest. Concentrations of 5-n-pentadecylresorcinol did not differ significantly across the three stages of ripening. The levels of these two resorcinols were found to be strongly inter-correlated (P<0.01, r²=0.71), with concentrations of 5-n-heptadecenylresorcinol being an average 18 times higher than those of 5-n-pentadecylresorcinol. At the ‘eating ripe’ stage, significant relationships were observed between the concentrations of each type of alk(en)ylresorcinol and anthracnose lesion areas following postharvest inoculation, P<0.001, r²= 0.69 for 5-n-pentadecylresorcinol, and P<0.001, r²= 0.44 for 5-n-heptadecenylresorcinol.     

Morphological evidences for onion-induced growth inhibition of Trichophyton rubrum and Trichophyton mentagrophytes

The antifungal activity of onion (Allium cepa L.) on two important dermatophytes, Trichophyton rubrum and Trichophyton mentagrophytes, with special reference to morphological aspects was studied. Growth of both fungi was found to be strongly inhibited by aqueous onion extract (AOE) as a dose-dependent manner. The extract showed fungicidal effect for both fungi at concentrations >3.12% (v/v). The fungus T. mentagrophytes was more affected by the onion as compared to T. rubrum at all concentrations used. Morphological effects of onion exposure were examined in correlation with fungal growth. Corresponding to the growth inhibition, light and electron microscopy observations revealed morphological anomalies in hyphal compartments. The results demonstrated that AOE targets the cell membrane of the fungi as breaking down of both inner and outer membranes with consequent extrution of materials into the surrounding medium. Cytoplasmic membranes and other membranous structures of organelles, such as nuclei and mitochondria, were also disrupted. In correlation to the fungal growth, morphological alterations occurred to a less content for T. rubrum compared with T. mentagrophytes. The hyphae of T. rubrum were found to be mainly affected by converting to resistant forms, i.e., chlamidospores as a consequence of phenotype switching response to AOE. Plasmolysis accompanied by an almost complete depletion and disorganization of cytoplasmic structures were found to be the final event which led to cell death. Ultrastructural evidences obtained from this study strongly support that morphological changes of T. rubrum and T. mentagrophytes caused by AOE are associated with its fungistatic and fungicidal activities. With respect to the morphological results and the preliminary data on fungal biochemistry, a mechanism of action by interacting of AOE with thiol (-SH) groups present in essential compartments of the fungal cells was postulated.     

Comparison of chitosans with different molecular weights as possible wood preservatives

Fungi cause serious problems in wood utilization, and environmentally benign wood protection is required as an alternative to traditional chemicals. Chitosan has shown promising antimicrobial properties against several microorganisms. In this study, we present the characterization of and antifungal properties of a commercial chitosan formulation developed for impregnation of wood. A broad range of chemical and mycological methods were used to evaluate the uptake, fixation, and antifungal properties of chitosan for wood preservation. The results show that the higher the uptake of chitosan the lower the relative recovery of chitosan in wood after leaching, and the higher the molecular weight of chitosan the higher the recovery. Chitosan with high molecular weight proved to be more efficient against decay fungi than chitosan with low molecular weight. The fungi tested on chitosan-amended nutrient agar medium were totally inhibited at 1% (w/v) concentration. In decay studies using small wood blocks, 4.8% (w/v) chitosan concentration gave the best protection against brown rot fungi.     

In vitro sensitivity of the amphibian pathogen Batrachochytrium dendrobatidis to antifungal therapeutics

Chytridiomycosis, a skin disease caused by Batrachochytrium dendrobatidis, has caused amphibian declines worldwide. Amphibians can be treated by percutaneous application of antimicrobials, but knowledge of in vitro susceptibility is lacking. Using a modified broth microdilution method, we describe the in vitro sensitivity of two Australian isolates of B. dendrobatidis to six antimicrobial agents. Growth inhibition was observed, by measurement of optical density, with all agents. Minimum inhibitory concentrations (µg/ml; isolate 1/2) were – voriconazole 0.016/0.008; itraconazole 0.032/0.016; terbinafine 0.063/0.063; fluconazole 0.31/0.31; chloramphenicol 12.5/12.5; amphotericin B 12.5/6.25. Killing effects on zoospores were assessed by observing motility. Amphotericin B and terbinafine killed zoospores within 5 and 30 min depending on concentration, but other antimicrobials were not effective at the highest concentrations tested (100 µg/ml). This knowledge will help in drug selection and treatment optimization. As terbinafine was potent and has rapid effects, study of its pharmacokinetics, safety and efficacy is recommended.     

GST-MtDef4融合蛋白的原核表达及体外抑菌活性的检测

为研究苜蓿(Medicago truncatula)防卫素基因MtDef4的抑菌功能,以含有该基因片段的质粒pAND-MtDef4为模板,扩增出相应的目的基因,测序正确后将MtDef4基因片段连接到带有GST标签的原核表达载体pGEX6P-1中,转化大肠杆菌TransB(DE3),经IPTG诱导,Glutathione Sepharose 4B亲和纯化,通过SDS-PAGE电泳及Western blot确定融合蛋白的表达。体外抑菌试验表明,MtDef4能够抑制病原真菌Corynespora cassiicola而非Collectotrichum gloeosporioides的生长。本研究结果可为进一步培育转基因橡胶抗病品种奠定基础。