Purification and infectivity ofEntomophaga grylli (Fresenius) Batko Pathotype 2 againstMelanoplus differentialis (Thomas) [Ort.: Locustidae]

The life stages ofEntomophaga grylli (Fresenius) Batko Pathotype 2 were purified and separated by centrifugation in Percoll^R density-gradient medium. The ranges of buoyant densities for germinated resting spores, germ conidia, and resting spores respectively were: 1.040–1.050, 1.055–1.085, and 1.080–1.120 g/ml. Cuticular invasion by germinated germ conidia was the means by whichMelanoplus grasshoppers became infected. Scanning electron micrographs revealed germination of germ conidia on the visible host integument at 100% RH, but not at 90% RH. Significantly higher mortality (P<0.05) was obtained after 3 weeks with grasshoppers incubated in constant light than in constant dark for 24 h following treatment. The disease was not transmitted by ingestion of any life stage. Contribution N^o 85-153-J, Department of Entomology. Agricultural Experiment Station, Kansas State University, Manhattan, KS 66506.     

Studies on the biosynthesis and metabolism of the phytoalexin lubimin and related compounds in Datura stramonium L.

Arachidonic acid, cellulase, CuSO₄, a sonicate of Phytophthora infestans mycelium and a spore suspension of Penicillium chrysogenum all elicited the formation of the sesquiterpenoid phytoalexins lubimin, 3-hydroxylubimin and rishitin in fruit cavities of Datura stramonium. 3-Hydroxylubimin was the predominant phytoalexin formed after treatment of the fruits with arachidonic acid, cellulase and the P. infestans preparation. Copper sulphate was a potent elicitor of lubimin but not 3-hydroxylubimin. The fungus P. chrysogenum metabolized lubimin and 3-hydroxylubimin to 15-dihydrolubimin and 3-hydroxy-15-dihydrolubimin respectively, both in fruit cavities inoculated with spores of this fungus and in pure culture. The 15-dihydrolubimin formed in the fruits by the fungus was further metabolized (by the fruits) to both isolubimin and 3-hydroxy-15-dihydrolubimin. The precursor-product relationships between all of the subject compounds was investigated by feeding experiments with ³H-labelled compounds. 2-Dehydro-[15-³H₁]lubimin was rapidly and efficiently incorporated into lubimin and may be the direct precursor of lubimin in planta. 3-Hydroxy[2-³H₁]lubimin was incorporated into the nor-eudesmane rishitin but 10-epi-3-hydroxy[2-³H₁]lubimin was not. An updated scheme for the biosynthesis and metabolism of lubimin and related compounds in infected tissues of solanaceous plants is presented.We thank Mr Vic Swetez for the provision of plant material, Mrs Margaret Huffee for technical assistance, Dr David Ewing for help with obtaining NMR spectra, and the Agricultural and Food Research Council for financial support.     

Effects of three herbicides on soil microbial biomass and activity

Three post-emergence herbicides (2,4-D, picloram and glyphosate) were applied to samples of an Alberta agricultural soil at concentrations of 0, 2, 20, and 200 μg g⁻¹. The effects of these chemicals on certain microbial variables was monitored over 27 days. All herbicides caused enhancement of basal respiration but only for 9 days following application, and only for concentrations of 200 μg g⁻¹. Substrate-induced respiration was temporarily depressed by 200 μg g⁻¹ picloram and 2,4-D, and briefly enhanced by 200 μg g⁻¹ glyphosate. It is concluded that because changes in microbial variables only occurred at herbicide concentrations of much higher than that which occurs following field application, the side-effects of these chemicals is probably of little ecological significance.     

Elicitor-induced metabolic changes in cell cultures of chickpea (Cicer arietinum L.) cultivars resistant and susceptible to Ascochyta rabiei

Cell-suspension cultures of two chickpea (Cicer arietinum L.) cultivars, resistant (ILC 3279) and susceptible (ILC 1929) to the fungus Ascochyta rabiei (Pass.) Lab., showed differential accumulation of the phytoalexins medicarpin and maackiain, and transient induction of related enzyme activities after application of an A. rabiei-derived elicitor. The chalcone-synthase (CHS) activity (EC 2.3.1.74) which is involved in the first part of phytoalexin biosynthesis exhibited a maximum 8–12 h after elicitation in the cells of both cultivars. Concomitant with the fivefold-higher phytoalexin accumulation, CHS activity increased twofold in the cells of the resistant cultivar. The maximum of the elicitor-induced CHS-mRNA activity was determined 4 h after onset of induction in the cultures of both cultivars, although in cells of cultivar ILC 3279 this mRNA activity was induced at a level twofold higher than that in cells of the susceptible race ILC 1929. Investigations of CHS isoenzymes by two-dimensional gel electrophoresis of immunoprecipitated in-vitro-translated protein indicated the presence of five proteins. In the cells of both cultivars only two of the isoenzymes were induced after elicitor treatment. Analysis of the total in-vitro-translated proteins by two-dimensional gel electrophoresis showed that the constitutively expressed patterns of mRNA activities in the cell cultures of the two cultivars were identical. After elicitation, considerably more translatable mRNAs were induced in the cells of cultivar ILC 3279. The few induced proteins, and their respective mRNA activities, which could be detected in the cells of the susceptible cultivar, all existed in the cells of the resistant cultivar, too. One highly induced protein (M_r 18 kDa) found in the cells of cultivar ILC 3279 reached its maximum mRNA activity 6 h after elicitor application. The amount of this protein was hardly increased in the cells of the susceptible cultivar. This protein appears to be excreted from the cells into the growth medium.Abbreviations CHS chalcone synthase - IEF isoelectric focussing - ILC international legume chickpea - PR-protein pathogenesis-related protein - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Financial support by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie is gratefully acknowledged. The authors thank Dr. K. Hahlbrock (Max-Planck-Institut für Züchtungsforschung, Köln, FRG) for provision of antisera and the International Centre for Agricultural Research in the Dry Areas (Aleppo, Syria) for plant material.     

Characterization of two class II chitinase genes from peanut and expression studies in transgenic tobacco plants

Two different genes encoding class II chitinases from peanut (Arachis hypogaea L. cv. NC4), A.h.Chi2;1 and A.h.Chi2;2, have been cloned. In peanut cell suspension cultures, mRNA levels of A.h.Chi2;2 increased after ethylene or salicylate treatment and in the presence of conidia from Botrytis cinerea. The second gene, A.h.Chi2;1, was only expressed after treatment with the fungal spores. Transgenic tobacco plants containing the complete peanut A.h.Chi2;1 gene exhibited essentially the same expression pattern in leaves as observed in peanut cell cultures. Expression characteristics of transgenic tobacco carrying a promoter-GUS fusion of A.h.Chi2;1 are described.     

Genotypic variation in zinc efficiency and resistance to crown rot disease (Fusarium graminearum Schw. Group 1) in wheat

A crown rot disease in wheat caused by the fungusFusarium graminearum Schw. Group 1 is a widespread problem in chronically Zn-deficient Australian soils. A link between crown rot and Zn deficiency was established by Sparrow and Graham (1988). This paper reports a test of a further hypothesis, that wheat genotypes more efficient at extracting zinc from low-zinc soils are more resistant to infection by this pathogen. Three wheat cultivars (Excalibur, Songlen and Durati) of differential Zn efficiency were tested at three zinc levels (0.05, 0.5 and 2.0 mg Zn kg⁻¹ of soil) and three levels ofF. graminearum S. Group 1 inoculum (0.1 g and 0.3 g kg⁻¹ live chaff-inoculum and control having 0.1 g kg⁻¹ dead chaff inoculum). Six weeks after sowing dry matter production of shoots and roots was decreased byFusarium inoculation at 0.05 mg and 0.5 mg kg⁻¹ applied Zn.Fusarium inoculum at 0.1 g was as effective as 0.3 g kg⁻¹ for infection and decreasing dry matter. The infection at the basal part of culm decreased significantly by increasing the rate of Zn application. Excalibur, a Zn-efficient cultivar (tolerant to Zn deficiency) produced significantly more shoot and root dry matter, and showed less disease infection compared with Zn-inefficient cultivars (Durati and Songlen) at low (0.05 mg Zn kg⁻¹ soil) and medium (0.5 mg Zn kg⁻¹ soil) Zn fertilization rates. Higher rate of Zn fertilization (2.0 mg Zn kg⁻¹ soil) reduced the disease level in Durati to the level of Excalibur but the disease level of Songlen was still high, indicating its high Zn requirement and or sensitivity to crown rot. The data on Zn uptake show that Excalibur, being Zn-efficient, was able to scavenge enough Zn from Zn-deficient soil, we suggest that besides sustaining growth Excalibur was able to build and maintain resistance to the pathogen; inefficient cultivars needed extra Zn fertilization to achieve performance comparable to that of Excalibur. The present study indicates that growing Zn-efficient cultivars of wheat along with judicious use of Zn fertilizer in Zn-deficient areas where crown rot is a problem may sustain wheat production by reducing the severity of the disease as well as by increasing the plant vigour through improved Zn nutrition. ei]Section editor: R Rodriques-Kalana     

Suppression of fungal β-glucan-induced plant defence in soybean (Glycine max L.) by cyclic 1,3-1,6-β-glucans from the symbiont Bradyrhizobium japonicum

The production of antimicrobial phytoalexins is one of the best-known inducible defence responses following microbial infection of plants or treatment with elicitors. In the legume soybean (Glycine max L.), 1,3-1,6--glucans derived from the fungal pathogen Phytophthora sojae have been identified as potent elicitors of the synthesis of the phytoalexin, glyceollin. Recently it has been reported that during symbiotic interaction between soybean and the nitrogen-fixing bacterium Bradyrhizobium japonicum USDA 110 the bacteria synthesize cyclic 1,3-1,6--glucans. Here we demonstrate that both the fungal and the bacterial -glucans are ligands of -glucan-binding sites which are putative receptors for the elicitor signal compounds in soybean roots. Whereas the fungal -glucans stimulate phytoalexin synthesis at low concentrations, the bacterial cyclic 1,3-1,6--glucans appear to be inactive even at relatively high concentrations. Competition studies indicate that increasing concentrations of the bacterial 1,3-1,6--glucans progressively inhibit stimulation of phytoalexin synthesis in a bioassay induced by the fungal 1,3-1,6--glucans. Another type of cyclic -glucan, a 1,2--glucan from Rhizobium meliloti, that does not nodulate on soybean, seems to be inactive as elicitor and as ligand of the -glucan-binding sites. These results may indicate a novel mechanism for a successful plant-symbiont interaction by suppressing the plant's defence response.Abbreviations HG-APEA 1-[2-(4-aminophenyl)ethyl]amino-l-[hexaglucosyl]deoxyglucitol - HG-AzPEA l-[2-(4-azidophenyl)-ethyl]amino-l-[hexaglucosyl]deoxyglucitol - IC₅₀ concentration for half-maximal displacement We thank Ines Arlt for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 369), the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie, Fonds der Chemischen Industrie (J.E.), and USDA CSRS NRI Competitive Research grant 93373059233 (A.A.B.).     

Identification and characterization of bacterial endophytes of rice

We isolated seven different bacteria from rice seedlings grown from surface sterilized seeds. Three were associated with the rice seed husk and the other four were growing endophytically within the seed. Microscopic studies revealed that the endophytes were concentrated in the root stele region. Some of the bacteria exhibited strong anti-fungal activity against Rhizoctonia solani, Pythium myriotylum, Guamannomyces graminis and Heterobasidium annosum.     

Paracoccidioides brasiliensis antigen batches from the same isolate show immunological and biochemical differences

We investigated the occurrence of antigenic and biochemical variability among Paracoccidioides brasiliensis antigen batches prepared according to the same protocol. Initially (experiment #1), we analyzed two antigen lots of two human isolates (Bt1 & Bt2), cultured in two media (PYG: bactopeptone, yeast extract, glucose; MMM: McVeigh & Morton medium) in SDS-PAGE and in two immunological tests (imunodiffusion-ID and footpad swelling test-FPT). Afterwards (experiment #2), we compared the antigenic profile of three antigen batches from three human isolates (Bt1, Bt2 & Bt3) by two-dimensional immunoelectrophoresis (2 D-IEP) against a reference system for P. brasiliensis antigens. In experiment #1, there were important intra- and inter-strain antigenic differences between batches of the fungal isolates cultured on both media. The block titration of the antigen batches for the immunological tests revealed correlation between protein concentration and biological activity in ID and no correlation in FPT. In experiment #2, the reference system for P. brasiliensis showed 26 antigen peaks. There were important differences between batches prepared from the same isolate and between batches from different isolates. Our data suggested the occurrence of instability in the synthesis of antigenic components by a same P. brasiliensis isolate, under controlled incubation conditions.