Growth inhibition of the cereal root pathogens Rhizoctonia solani AG8, R. oryzae and Gaeumannomyces graminis var. tritici by a recombinant 42-kDa endochitinase from Trichoderma harzianum

The objective of this study was to determine the effectiveness of a 42-kDa endochitinase coded by the ThEn42 gene from Trichoderma harzianum as a potential source of transgenic resistance to Rhizoctonia root rot of barley caused by Rhizoctonia solani AG8 and/or R. oryzae. The gene cThEn42 was codon optimized (GC content increased from 53.3 to 65.1%) and then synthesized to produce the modified cThEn42GC in Pichia pastoris for in vitro tests. Two expression vectors were constructed: one with the fungal signal peptide and the fungal activation peptide [FSP-FAP-cThEn(GC)] and the other with barley chitinase 26 signal peptide followed by the fungal signal and activation peptides [SP(HVChi26)-FSP-FAP-cThEn(GC)]. N-terminal sequencing showed that, of two proteins secreted into liquid medium, FSP was cleaved off faithfully in one protein and both FSP and FAP were cleaved from the other protein. Purified endochitinase provided strong in vitro inhibition of both R. solani AG8 and R. oryzae. The enzyme had an intermediate inhibitory activity against Gaeumannomyces graminis var. tritici, and no inhibitory activity against Fusarium graminearum, F. pseudograminearum, and F. culmorum.     

The potential of non-pathogenic Gaeumannomyces spp., occurring naturally or introduced into wheat crops or preceding crops, for controlling take-all in wheat

Take‐all disease (Gaeumannomyces graminis var. tritici) in wheat crops is known to be suppressed by naturally occurring antagonistic fungi, closely related to the pathogen, that infect grasses and cereals. This form of suppression was re‐investigated because of the changing importance and role of grass weeds and grass covers in arable farming. Natural populations of the competitive fungus Gaeumannomyces cylindrosporus, allowed to develop under rye‐grass, were more effective than artificially introduced populations in suppressing the development of take‐all in following wheat crops. To be effective, the antagonist needs to be present before the start of wheat cropping. Introducing G. cylindrosporus, but not G. graminis var. graminis (a potential antagonist that is faster growing), into a previous crop, or just after the previous crop, sometimes suppressed take‐all, but the effect was small. It is concluded that, for any future attempts at biocontrol by these fungi, they should be introduced into a preceding crop not susceptible to take‐all. Take‐all inoculum in the soil should be at a minimum and effective hosts of the take‐all pathogen must not be present as weeds or volunteers.     

Field tests of bacteria and soil-applied fungicides as control agents for take-all in winter wheat

Putative biological and chemical treatments for controlling take-all were used in each of three consecutive years at two locations where winter wheat crops were grown in naturally-infested fields. The chemical treatments more often decreased take-all than the biological treatments, but no treatment consistently and significantly decreased take-all, nor did any cause a significant increase in yield. An isolate of Bacillus cereus var. mycoides and one of B. pumilis, applied as soil drenches in autumn or spring, or in the seed furrows, were usually ineffective. Of the few significant effects on disease, half were associated with increases and half with decreases, and most occurred in April and did not persist to late June. Two strains of Pseudomonas pluorescens applied to the seed were ineffective. The fungicide benomyl, applied as a drench in autumn and spring at 20 kg/ha was ineffective, while nuarimol, applied as a drench in autumn at 2 kg/ha was sometimes effective. Nuarimol incorporated into the seed bed at 2 kg/ha was the most effective treatment. In analyses using a functional relationship model for data from treated and untreated plots 12% of 176 data sets for biological treatments, 38% of 96 data sets for chemical treatments and 81% of 16 data sets for combined treatments showed increasing efficiency of the treatment with increasing disease intensity. These findings also demonstrate an additional advantage of the experimental design, namely that treatments are tested at different disease intensity levels within fields.     

Effect of certain herbicide treatments on pasture composition and inoculum of the take-all fungus

The improvement of pastures by the use of a range of herbicides to eliminate grasses, and their effect on populations of the take-all fungus (Gaeumannomyces graminis vartritici=Ggt) were studied in the field (at Esperance Downs, on the south-coast of Western Australia) from 1982 to 1985. Field trials were conducted to evaluate three herbicide treatments (2,4-D amine+propyzamide; 2,4-D amine+paraquat; paraquat/ diquat) and an unsprayed control. A pot trial involving these treatments with two levels of nitrogen was undertaken to confirm treatment effects observed in the field trial. All herbicide treatments resulted in reduced grass composition of pastures, in both the year of spraying and in the second year of pasture, but reduced dry matter production in the year of spraying. In the year of spraying, however, inoculum ofGgt was reduced (P<0.1) only following the 2,4-D amine+propyzamide treatment and was greater (P<0.1) after 2,4-D amine+paraquat treatment than the unsprayed treatment. Despite reduced grass levels in the herbicide-treated plots in the second year of pasture,Ggt inoculum did not differ between treatments, nor did it after a wheat crop which followed a second year pasture. There was high correlation (P<0.001) between disease levels and dry weights of grasses in the pot trial. There was significantly less (P<0.001) grass in pots treated with herbicides compared to the unsprayed control but no difference (P>0.05) was evident between treatments. Inoculum levels were lower (P<0.05) in the treated pots than the unsprayed control with no evidence of differences among treatments (P>0.05). Nitrogen level had no effect on disease (P>0.05). All herbicide treatments tested reduced grass level and total dry matter, both in the field and in pots. Whereas in the pot trial reduced grass levels resulted in reducedGgt inoculum, in the field such a reduction occurred only with the 2,4-D amine+propyzamide treatment and only in the year of spraying. Herbicide treatments had no effect onGgt inoculum in second year of pasture or crop. Unknown soil and environmental factors in the field precluded a simple relationship between grass level in pasture and subsequent level ofGgt inoculum, and where such a relationship did occur (2,4-D amine+propyzamide treatment) it appeared to be shortlived.     

Pichia expression and mutagenesis of 7,8-linoleate diol synthase change the dioxygenase and hydroperoxide isomerase

Linoleate diol synthases (LDS) are homologous 8(R)-dioxygenases with hydroperoxide isomerase activities, expressed in fungal pathogens of humanitarian importance. We report for the first time expression and site-directed mutagenesis of LDS. 7,8-LDS of the take-all fungus, expressed in Pichia pastoris, oxygenated 18:2n − 6 to 8(R)-hydroperoxylinoleic acid, which was unexpectedly isomerized to 5,8(R)-dihydroxylinoleic acid (60% 5S) and to 8(R),13-dihydroxyoctadeca-9(E),11(E)-dienoic acid. The latter was likely formed via hydrolysis of an unstable intermediate, 8(R),9(S)-epoxyoctadeca-10(E),12(Z)-dienoic acid. A tyrosyl radical is formed during 7,8-LDS catalysis, and Tyr376 is the sequence homolog to Tyr385 of cyclooxygenase-1. Tyr376Phe retained hydroperoxide isomerase activity but lacked 8(R)-dioxygenase activity. The putative proximal heme ligand His379 and the N-glycosylation site at Asn216 appeared to be critical for 8(R)-dioxygenase activity, as His379Gln and Asn216Gln were inactive. Treatment with α-mannosidase to shorten N- and O-linked mannosides inhibited the hydroperoxide isomerase but not the 8(R)-dioxygenase. Our results suggest that post-translational modifications may influence the oxidation mechanism of 7,8-LDS.     

小麦全蚀病菌原生质体制备的条件及再生菌株的致病性

分别利用崩溃酶、溶壁酶、纤维素酶和蜗牛酶酶解小麦全蚀病菌,进行原生质体的制备试验。结果显示,4种酶均能消化该菌细胞壁,获得一定数量的原生质体;产生原生质体效率最高的是溶壁酶,该酶在浓度为16 mg/mL时产生的原生质体数量最多,最佳的酶解时间为2~3 h,最适作用温度为28℃。制备的原生质体可以再生并与原始出发菌株具有相同的致病能力。     

Immunolocalization of 1,3‐β‐Glucanases Secreted by Gaeumannomyces graminis var. tritici in Infected Wheat Roots

The distribution of extracellular 1,3‐β‐glucanase secreted by Gaeumannomyces graminis var. tritici (Ggt) was investigated in situ in inoculated wheat roots by immunogold labelling and transmission electron microscopy. Antiserum was prepared by subcutaneously injecting rabbits with purified 1,3‐β‐glucanase secreted by the pathogenic fungus. A specific antibody of 1,3‐β‐glucanase, anti‐GluGgt, was purified and characterized. Double immunodiffusion tests revealed that the antiserum was specific for 1,3‐β‐glucanase of Ggt, but not for 1,3‐β‐glucanase from wheat plants. Native polyacrylamide gel electrophoresis of the purified and crude enzyme extract and immunoblotting showed that the antibody was monospecific for 1,3‐β‐glucanase in fungal extracellular protein populations. After incubation of ultrathin sections of pathogen‐infected wheat roots with anti‐1,3‐β‐glucanase antibody and the secondary antibody, deposition of gold particles occurred over hyphal cells and the host tissue. Hyphal cell walls and septa as well as membranous structures showed regular labelling with gold particles, while few gold particles were detected over the cytoplasm and other organelles such as mitochondria and vacuoles. In host tissues, cell walls in contact with the hyphae usually exhibited a few gold particles, whereas host cytoplasm and cell walls distant from the hyphae were free of labelling. Furthermore, over lignitubers in the infected host cells labelling with gold particles was detected. No gold particles were found over sections of non‐inoculated wheat roots. The results indicate that 1,3‐β‐glucanase secreted by Ggt may be involved in pathogenesis of the take‐all fungus through degradation of callose in postinfectionally formed cell wall appositions, such as lignitubers.     

Colonisation of barley roots by endophytic Fusarium equiseti and Pochonia chlamydosporia: Effects on plant growth and disease

Colonisation of plant roots by endophytic fungi may confer benefits to the host such as protection against abiotic or biotic stresses or plant growth promotion. The exploitation of these properties is of great relevance at an applied level, either to increase yields of agricultural crops or in reforestation activities. Fusarium equiseti is a naturally occurring endophyte in vegetation under stress in Mediterranean ecosystems. Pochonia chlamydosporia is a nematode egg-parasitic fungus with a worldwide distribution. Both fungi have the capacity to colonise roots of non-host plants endophytically and to protect them against phytopathogenic fungi under laboratory conditions. The aim of this study was to evaluate the root population dynamics of these fungi under non-axenic practical conditions. Both fungal species were inoculated into barley roots. Their presence in roots and effects on plant growth and incidence of disease caused by the pathogen Gaeumannomyces graminis var. tritici were monitored periodically. Both fungi colonised barley roots endophytically over the duration of the experiment and competed with other existing fungal root colonisers. Furthermore, colonisation of roots by P. chlamydosporia promoted plant growth. Although a clear suppressive effect on disease could not be detected, F. equiseti isolates reduced the mean root lesion length caused by the pathogen. Results of this work suggest that both F. equiseti and P. chlamydosporia are long-term root endophytes that confer beneficial effects to the host plant.