The toxicity of Bacillus thuringiensis is temperature sensitive. Incubation of Caenorhabditis elegans with nematicidal B. thuringiensis strains at 16, 20, and 25 degrees C shows that toxicity decreases as temperature declines. At 16 degrees C, toxicity is completely lost, while it is maximal at 25 degrees C. Toxicity is pH sensitive and is significantly reduced when nematodes are incubated with the weak bases NH4Cl, chloroquine, acridine orange, methyl red, and neutral red. Based on these results, we proposed the hypothesis that the nematicidal factor is effectively internalized into the intestinal cells, a sharp deviation from the insecticidal B. thuringiensis toxins acting at the level of the brush border membrane. Although the absence of purified toxin prevents a more definitive elucidation of the mode of action, the results of this third and final part of this series of publications convincingly indicate that nematicidal B. thuringiensis do not hold the same promise as a biological control agent as the insecticidal B. thuringiensis strains.

Light-microscopic observations of the toxic action of Bacillus thuringiensis spore/crystals reveals that, in Caenorhabditis elegans, the intestine is destroyed in two stages over a period of 24 h. The anterior ring of four cells is the first and foremost target. Observations indicate that the intestine is the only tissue being destroyed. Screening of fourteen additional rhabditid nematode species against three nematicidal B. thuringiensis strains active against C. elegans, resulted in only one additional sensitive nematode species, and indicates a high species specificity of the nematicidal factor. However, in contrast to insect-specific B. thuringiensis toxins, the nematicidal toxin exhibits low developmental stage specificity against C. elegans; all developmental stages, including adult nematodes are sensitive. Moreover, sensitivity increases as development proceeds. Using ethyl methyl sulfonate induced mutagenesis two mutants of C. elegans have been recovered, exhibiting reduced sensitivity of up to 50 % against one of the nematicidal strains. Moreover, one of the mutants exhibited cross-resistance to a second nematicidal B. thuringiensis strain against which it was not screened. Preliminary data indicate that the reduced sensitivity in the mutants is not due to reduced pharyngeal pumping activity.

TransrrUssion electron rrUcroscopy is used to describe the intoxication in CaenorhabdiLis elegans, feeding on toxic spore/crystals of Bacillus thuringiensis. The toxin acts directly against the intestine, fLrst by affecting the anteriormost ring of four intestinal cells. Over a period of 12 hours, these cells lose much of their volume, the microvilli regress slowly, several cell organelles undergo dramatic change and are ultimately destroyed. No ruprure of the apical intestinal cell membrane is observed. Non-intestinal tissues seem unaffected. This srudy indicates considerable ultrastrucrural differences in the mode of action bet\veen the nematicidal toxin and the insecticidal crystal toxins from B. thun'ngiensis.

During screening of Bacillus thuringiensis isolates for nematicidal activity it was observed that spores of B. thuringiensis germinated in the intestine of bacteriophagous nematodes in the presence of antibiotics. This phenomenon was studied more closely by scanning electron microscopy. The nematodes were fed with bacterial spore-crystal mixtures in axenic culture medium supplemented with tetracyclin and chloramphenicol. Germination of spores was rare but was more frequently observed in Panagrellus redivivus than in other nematode species investigated. Germination of spores in the nematode intestine resulted in the colonization of the entire nematode within 24 hr. Crude nematode tissue preparations supported germination and subsequent growth of B. thuringiensis spores and vegetative cells. The mechanism for the loss of antibiotic activity in the nematode intestine is unknown. Since B. thuringiensis requires a nutrient-rich environment for reproduction, e.g., a cadaver, bacteriophagous nematodes may serve as suitable hosts for B. thuringiensis.

The nematicidal activity of the spore-crystal mixtures of three Bacillus thuringiensis isolates against hatched juveniles and adults of Caenorhabditis elegans was investigated. Toxicity was determined by adding 50-mu l aliquots of the spore-crystal mixtures to microtitre plate wells containing 50-mu l aqueous suspensions of 200-400 hatched juveniles and adults of C. elegans. Nematode mortality was observed from 8 hours incubation onwards; after 24 hours incubation no more significant increases in nematode mortality occurred. Nematode mortality varied from about 50 to 60 % when the nematicidal activity was tested in distilled water and was usually somewhat higher (but less than 10 %) when tested in axenic medium. Toxicity varied between the three isolates. Concentrations of at least 10(8) particles/ml were necessary to cause a nematode mortality higher than 30 %. Nematicidal activity was only observed when spore-crystal mixtures from at least 2-day-old cultures, consisting of about 50 % of vegetative cells, often containing a spore, and for about 50 % of a mixture of spores and crystals, were used. Heating to 75 degrees C and higher for 24 hours and autoclaving at 120 degrees C for 20 min destroyed the nematicidal activity of all three isolates. Differences in stability of the nematicidal activity were observed between the three isolates. In two isolates the nematicidal activity did not decline after storage at 28 degrees C for 15 days; in the third isolate the nematicidal activity declined after storage at 28 degrees C for 7 days. Multiple freezing at -20 degrees C or -70 degrees C and thawing had no effect on the nematicidal activity of two isolates but decreased the nematicidal activity of the third isolate. pH changes resulted in differences in stability of the nematicidal activity between the three isolates. These results may indicate the presence of different toxins.

Rhizomes of stinging nettle contain a small-sized lectin that exhibits binding specificity toward chitin. This lectin inhibits growth of several phytopathogenic and saprophytic chitin-containing fungi in vitro. The antifungal action of the nettle lectin differs from the action of chitinases, which are a ubiquitous class of antifungal plant proteins. Moreover, the nettle lectin acts synergistically with chitinase in inhibiting fungal growth. The nettle lectin may be a promising candidate for possible applications in the genetic engineering of disease-resistant crops.

AbstractExtensive studies showed that no disease was caused when seeds of different forage grasses were inoculated with Xanthomonas campestris pv. graminis. The disease could easily be induced by infecting the plants in the root system, leaves or flower. The inoculation site in the leaf proved to be of vital importance for the development of the disease. Wilting symptoms were quickly induced when the pathogen was inoculated near the leaf base. Plants in root‐contact with diseased plants showed disease symptoms. It is not known whether these symptoms were caused by the bacteria or by toxins released by nearby diseased plants. Cross inoculation trials on different grass varieties revealed that different pathovars exist in the group of xanthomonads, pathogenic to forage grasses. Some have a broad host range whereas others are more limited to a single plant genus. Field trials suggest that in Belgian climatic conditions, the losses caused by bacterial wilt are rather limited.

AbstractThe effect of host‐plant age, environmental humidity and temperature on the development of the wilting disease of Lolium perenne L., caused by Xanthomonas campestris pv. graminis, was examined. Very young plants were more resistant than older ones. Dry and warm conditions favoured the development and expression of the disease symptoms. Losses in yield were observed under several environmental conditions.

Antifungal rhizobacteria were obtained from maize, barley and chicory using direct or indirect isolation procedures. Effective isolates were tested for broad‐spectrum activity against a set of phytopathogenic fungi. Isolates with broad‐spectrum activity were identified as Pseudomonas fluorescens, P. cepacia, Serratia liquefaciens, S. plymuthica and Bacillus sp. Broad‐spectrum compounds produced by P. cepacia and Erwinia herbicola were characterized as pyrrolnitrin and herbicolin‐like compounds respectively.

During the growing season of 1984, the rhizobacteria (including organisms from the rhizosphere soil, the rhizoplane, and internal root zones) of 47 maize plants (two varieties) sampled from different locations in France and at different growth stages were inventoried. Isolates were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of their total cell proteins and were found to represent 352 different protein electrotypes. Maize seedlings were initially colonized by a small number of different strains. Densities reached up to 10 8 CFU/g of root. Later in the season, the population density decreased but the heterogeneity of the rhizobacterial populations increased. Fluorescent pseudomonads represented up to 35% of the total rhizobacterial population and comprised 43 different electrotypes. Other bacteria regularly present were Xanthomonas maltophilia, Serratia liquefaciens, Pseudomonas paucimobilis, and Bacillus spp. There was a very low similarity between rhizobacterial populations of plants of the same cultivar (LG5) within one field at different growth stages and also between rhizobacterial populations of the cultivars LG5 and BRIO42 on the same field. Most electrotypes (76%) were found on a single occasion. None of the 352 electrotypes was present on all plants. In the 1985 analysis the rhizobacteria of maize seedlings (one variety) sampled from one field were characterized. They represented 236 different protein electrotypes. Thirty-three isolates showed antifungal activity against major maize pathogens; they comprised four Pseudomonas cepacia strains, producing pyrrolnitrin as well as another unknown antifungal compound.

AbstractFive bacterial isolates were obtained in a nursery near Gent (Belgium) from diseased Dieffenbachia maculata (Lodd.) G., Don. cv. Compacta, cv. Camillo and cv. Veerle plants. They were identified by API 20E, API 50 CHE and API ZYM systems as Erwinia chrysanthemi. These strains and seven collection strains were pathogenic to all three D. maculata cultivars tested: cv. Camillo, cv. Compacta and cv. Tropic Snow. Inoculation in the stem or petiole was the only effective method for obtaining systemic infection. The petiole appeared to be the part of the plant in which disease developed most readily. Wounding was required for the induction of the disease. Temperatures between 25° and 30° C, a relative air humidity of 75 % or more and low light intensity (0.5 ft‐c) favoured the disease, whereas high inoculum sizes (106 or more cells) accelerated and increased it. Histological studies showed tissue degradation in infected areas. Petioles were most severely affected. Transport of bacteria seemed to occur after vessel infection in stem or petiole tissues.