Infection Biology of Bacterial Wilt of Forage Grasses

Infection of perennial ryegrass (Lolium perenne L.), Italian ryegrass (Loliummultiflorum L.), and timothy (Phleum pratense L.) by Xanthomonas campestris pv. graminis and Xanthomonas campestris pv. phlei occurred mainly via wounds rather than natural openings. Nevertheless, bacteria were detected by isolation and immunofluorescence in plants sprayed with the pathogen without prior wounding and in plants in which intact ears had been dipped in inocula. High concentrations of bacteria were observed around the stomata of perennial ryegrass and timothy by scanning electron microscopy 48 h after inoculation. Perennial ryegrass and Italian ryegrass and individual plants of ryegrass and timothy differedin susceptibility to the pathogens.     

Understanding the mechanism of biological control of passionfruit bacterial blight promoted by autochthonous phylloplane bacteria

The knowledge of biological control mechanism that provides a significant reduction in disease severity can guide the screening procedures and allow the discovery of new possibilities for disease control. In this study, a reduction of bacterial blight severity, caused by three Xanthomonas axonopodis pv. passiflorae (Xap) strains, was demonstrated by using nine indigenous passionfruit phylloplane bacteria regardless of the pathogen origin. Experiments were done to elucidate preemptive exclusion through carbon sources utilization profiles, siderophores production, iron competition and antibiosis. Furthermore, peroxidase activity and spatial separation assays were conducted to evaluate the ability to induce systemic resistance. Identification by 16S rRNA gene sequencing revealed that nine phylloplane strains show highest similarity to Arthrobacter, Curtobacterium, Enterobacter, Microbacterium, Pseudomonas or Stenotrophomonas. It is concluded that competition for iron and nitrogenate compounds on leaves explains the ability of bacterial phylloplane antagonists to control the disease. For siderophore-producing organisms that present antibiosis evidence, complementary iron supplementation assay is mandatory, to avoid misinterpretation. Results suggest that lack of these compounds could limit the optimal conditions for phylloplane colonization by Xap along the infection process.     

Phylloplane bacteria increase the negative impact of food limitation on insect fitness

1. When populations of herbivorous insects increase in density, they can alter the quantity or quality of their food. The impacts of diet‐related stressors on insect fitness have been investigated singly, but not simultaneously. 2. Foliage quantity and quality of red alder, Alnus rubra, were manipulated together with the presence of non‐entomopathogenic phylloplane bacteria to investigate their impacts, singly and in combination, on survival, pupal mass, growth rate, fecundity and egg quality of a cyclic forest insect, the western tent caterpillar, Malacosoma californicum pluviale. 3. Food limitation (half food) had strong negative impacts on all life‐history traits. When the larvae were fed continuously, however, neither ingesting phylloplane bacteria nor eating leaves from damaged branches (induced foliage) affected survival. In the half‐food treatment, ingesting bacteria further increased mortality, while feeding on induced foliage improved survival. 4. Growth rate and pupal mass of both sexes were reduced for larvae with food limitation compared with continuously fed insects and this was exacerbated when the larvae also ate bacteria‐treated leaves. A combination of bacteria and induced foliage also reduced larval growth rate by 5% in the full‐food treatment. 5. Fecundity (eggs per egg mass) was 2.7 times greater in full‐food than in food‐limited treatments but neither phylloplane bacteria nor plant induction had an effect. Insects fed induced foliage produced smaller eggs. Overall, there was no evidence of a three‐way interaction between the three stressors, although there were negative synergistic effects, primarily between food limitation and the ingestion of phylloplane bacteria.     

Olive Knot Pathogen with Pronounced Epiphytic Lifestyle is not Present in Association to Leaf Surface of European Olive Across the Himalayas in Nepal

Pseudomonas savastanoi pv. savastanoi, the causal agent of olive knot disease, often switches from a saprophytic to a parasitic lifestyle only following natural or man‐made activities, that cause lesions on plant tissues. We investigated the possible presence of this pathogen on the phylloplane of 28 Italian olive cultivars, recently introduced to Nepal. Although a consistent number of bacterial species were found in association with leaf, there was no presence of olive knot pathogen across the study area. The results suggest that the introduction of a new plant species in a given area does not necessarily introduce the pathogen when the propagation materials are rigorously supervised for pathogen exclusion.     

Triterpeneglycosides as Inhibitors of Fungal Growth and Metabolism

A venacin, the resistance factor in oat roots against Ophio-bolus graminis var. graminis, and a related triterpeneglycoside, aescin, induced a rapid release of K⁺ from mycelia of Opbio-bolus graminis and Neurospora crassa, suspended in phosphate buffer. N. crassa also released Mg²⁺ whereas no outflux of Mg²⁺ was found from O. graminis. The inhibitors induced a release of inorganic phosphate into acetate buffer from Neurospora crassa. The amount of inorganic phosphate in the mycelia decreased when O. graminis and N. crassa were treated with the inhibitors in phosphate buffer. In other media the inhibitors had weak or no effects on the ion contents of the mycelia. The effect of aescin was low in Aspergillus niger and nil in Pythium irregulare. However, high amounts of K⁺, Mg²⁺, and phosphate ions were lost to the medium when the mycelium of P. irregulare, washed with distilled water, was suspended in different buffers. The ions lost were reabsorbed during the experimental period. The leakage of ions indicates that the plasma membrane of the growth sensitive fungi is severely affected by the inhibitors, while a corresponding effect on the growth insensitive fungi does not take place.     

Effects of Species Diversity on Establishment and Coexistence: A Phylloplane Fungal Community Model System

A model system was devised, evaluating the influence that species diversity (species richness) has on fungal establishment and coexistence. Seven members of the fungal phylloplane community of Vaccinium macrocarpon (American cranberry) were selected to assess how species diversity affected development and coexistence of another community member, Pestalotia vaccinii. Pestalotia was engaged in competitive interactions on 1% Malt Extract Agar (MEA) petri dishes with each of the seven individual saprotrophs (two-way interaction), in random combinations with three of the seven saprotrophs (four-way interaction), and in random combinations with five of the seven saprotrophs (six-way interaction). The saprotrophic fungi used in this study were Aspergillus sp., Alternaria alternata, Cladosporium cladosporoides, Curvularia lunata, Epicoccum purpuracens, Penicillium sp., and Pithomyces chartarum. We hypothesized that species diversity would have a significant impact on the establishment and coexistence of Pestalotia vaccinii in culture. In an effort to minimize density-dependent effects, the number of viable spores employed in the three types of interactions was kept constant. Target spore concentrations of 50 viable spores of P. vaccinii and 50 saprotroph spores were used, regardless of the number of species involved in the interaction. This proved to be a very important factor in the experiment. As our results show, species diversity had little or no effect on the establishment and coexistence of Pestalotia vaccinii; however, spore density played an extremely important role in the establishment and development of fungal propagules in our model.     

核桃黑斑病拮抗放线菌WMF106的筛选、鉴定及防效

【背景】核桃黑斑病是由2种病原菌引起的细菌性病害,目前缺乏有效的生物防治方法。【目的】从核桃树根际土壤中筛选对核桃黑斑病病原菌具有拮抗效果的放线菌菌株,为该病害生防菌剂的开发提供基础。【方法】采用稀释涂布法分离放线菌,并以病原菌野油菜黄单胞菌(Xanthomonas campestris pv. campestris)和成团泛菌(Pantoea agglomerans)作为指示菌,利用平板对峙法和改良牛津杯法筛选具有高拮抗活性的菌株,通过形态学特征、生理生化特性和16SrRNA基因序列分析确定其分类地位,并测定其无菌发酵液的抗菌谱和室内防效。【结果】筛选到一株对野油菜黄单胞菌和成团泛菌均有较强拮抗作用的放线菌菌株WMF106,该菌株对2种病原菌的抑菌圈直径分别为2.38 cm和1.82 cm,无菌发酵液对2种病原菌的抑菌圈直径分别为1.75 cm和1.55 cm。根据菌株形态学、生理生化特性及16SrRNA基因序列分析,将菌株WMF106鉴定为暗蓝色链霉菌(Streptomyces caeruleatus)。该菌株对尖孢镰刀菌、腐皮镰孢菌、辣椒刺盘孢菌、灰葡萄孢菌、胶孢炭疽菌5种植物病原菌及大肠杆菌、金黄色葡萄球菌、铜绿假单胞菌、白色念珠菌、黑曲霉5种指示菌均有抑制作用,抗菌性能广谱高效,其无菌发酵液原液对离体叶片上由野油菜黄单胞菌和成团泛菌造成的核桃黑斑病防效分别为77.44%和58.33%。【结论】菌株WMF106可作为防治核桃黑斑病的生防材料,具有良好的开发价值和应用前景。     

Biological control of winter wheat pathogens with the use of antagonistic Sphingomonas bacteria under greenhouse conditions

This paper describes, for the first time, the effect of bacteria of the genus Sphingomonas on healthiness of winter wheat. The effect of the application of Sphingomonas cell suspension on development of disease symptoms of powdery mildew and Fusarium head blight (FHB) of winter wheat cv. Bogatka was studied under greenhouse conditions. The abundance of populations of yeast and fungi producing mycelium as well as bacteria of the genus Azotobacter and pseudomonads was determined on wheat kernels. The biocontrol agent reduced the population size of Fusarium poae, and it contributed to better grain filling. The tested Sphingomonas isolate reduced the severity of flag leaf infection caused by pathogenic biotroph Blumeria graminis f. sp. tritici.     

Parasites of European hedgehogs (<Emphasis Type="BoldItalic">Erinaceus europaeus</Emphasis>) in Britain: epidemiological study and coprological test evaluation

Seventy-four European hedgehogs (Erinaceus europaeus) that had died in wildlife rehabilitation centres were dissected and their parasite burdens documented. Overall parasite prevalence was 91%, and a total of six helminth species were isolated: five nematodes (Crenosoma striatum, Eucoleus aerophilus, Capillaria erinacei, Capillaria ovoreticulata and Capillaria spp.), one trematode (Brachylaemus erinacei) and one acanthocephalan (Oliganthorhynchus erinacei). The tick Ixodes hexagonus and flea Archeopsylla erinacei were also collected. The effect of parasite infection on body condition was assessed by correlation of burdens with the residuals of weight–skeletal length regression. Tick presence was positively related to body condition; for other parasites, no significant relationship was found. Faecal egg or larval count was closely correlated with adult parasite burden for C. striatum and Capillaria/Eucoleus spp., but not for other species. Coprological analysis should therefore be useful for in vivo studies of nematode parasite infection in hedgehogs. The epidemiology of parasites in hedgehogs and their possible role in recent population declines are discussed.     

101 Effect of Karlodinium Micrum Toxin on Success of the Parasitic Dinoflagellate Amoebophyra SP.

Parasitic dinoflagellates of the genus Amoebophrya infect and kill bloom‐forming dinoflagellates, including the toxic species Karlodinium micrum. Unlike non‐toxic hosts, K. micrum is partially resistant to infection, a trait that may be related to toxin production. Here we tested the hypothesis that parasitism of K. micrum is inversely related to toxin concentration in the culture medium. Time‐course studies were conducted to determine the influence of extracted toxin and toxin carrier (methanol) on host growth, parasite prevalence, and parasite load. Results indicate that methanol concentrations below 0.1% have no effect on these variables. When methanol concentration was maintained below 0.1%, extracted toxin equivalent to 100 to 10,000 K. micrum per ml had no effect on host abundance. We are currently analyzing sample to assess the fate of Amoebophrya dinospores when exposed to K. micrum toxin. We will also consider the effect of intracellular host toxin on parasite success, by examining the fate of Amoebophrya dinospores when inoculated to K. micrum cultures that express different levels of toxin production. Understanding the effect of toxins on parasite success will contribute to our knowledge of host‐parasite biology and provide insight into the role of dinoflagellate toxins as a defense against parasitism.     

A new bacteria-free strategy induced by MaGal2 facilitates pinewood nematode escape immune response from its vector beetle

Symbiotic microbes play a crucial role in regulating parasite–host interactions; however, the role of bacterial associates in parasite–host interactions requires elucidation. In this study, we showed that, instead of introducing numerous symbiotic bacteria, dispersal of 4th-stage juvenile (J_IV) pinewood nematodes (PWNs), Bursaphelenchus xylophilus, only introduced few bacteria to its vector beetle, Monochamus alternatus (Ma). J_IV showed weak binding ability to five dominant bacteria species isolated from the beetles’ pupal chamber. This was especially the case for binding to the opportunistic pathogenic species Serratia marcescens; the nematodes’ bacteria binding ability at this critical stage when it infiltrates Ma for dispersal was much weaker compared with Caenorhabditis elegans, Diplogasteroides asiaticus, and propagative-stage PWN. The associated bacterium S. marcescens, which was isolated from the beetles’ pupal chambers, was unfavorable to Ma, because it caused a higher mortality rate upon injection into tracheae. In addition, S. marcescens in the tracheae caused more immune effector disorders compared with PWN alone. Ma_Galectin2 (MaGal2), a pattern-recognition receptor, was up-regulated following PWN loading. Recombinant MaGal2 protein formed aggregates with five dominant associated bacteria in vitro. Moreover, MaGal2 knockdown beetles had up-regulated prophenoloxidase gene expression, increased phenoloxidase activity, and decreased PWN loading. Our study revealed a previously unknown strategy for immune evasion of this plant pathogen inside its vector, and provides novel insights into the role of bacteria in parasite–host interactions.     

Multispecies interactions among a plant parasite,a pollinator and a seed predator affect the reproductive output of an invasive plant,Cytisus scoparius

Interactions between species pairs are almost always mediated by interactions with other species. The outcomes of these multispecies interactions are often difficult to predict and are rarely studied. In addition to their theoretical importance, multispecies interactions are also important for management situations. Where multiple agents are used to control invasive species, interactions between agents may either enhance or reduce the impacts on the target species, or may simply have additive effects. In this study, conducted in a Mediterranean‐type woodland in Australia, we examined how the interaction between an invasive legume, Cytisus scoparius (Leguminosae), its pollinator, Apis mellifera and a seed predator, Bruchidius villosus (Bruchidae), are modified by a native hemiparasitic vine, Cassytha pubescens (Loranthaceae). The parasite had a direct negative effect on C. scoparius, reducing flowering by 50% and consequently fruit and seed production. Despite having fewer flowers, infected plants had the same proportion of ‘tripped’ flowers, an indirect measure of pollinator visitation, as uninfected plants; although fruit formed on infected plants it was more likely to abort prematurely. Seed predation by B. villosus was lower on parasite‐infected C. scoparius plants than in uninfected plants. Although Ca. pubescens had an antagonistic effect on B. villosus, in consort, the two agents reduced overall seed production by an average of 62%. The acquired parasite Ca. pubescens was more effective in reducing reproductive output than the introduced seed predator, B. villosus, and shows potential as a biocontrol agent for C. scoparius. We documented a subadditive effect of two biological enemies on the invasive species, where the acquired parasite had a stronger effect than the introduced seed predator.     

Differential impacts of shared parasites on fitness components among competing hosts

Effects of parasites on individual hosts can eventually translate to impacts on host communities. In particular, parasitism can differentially affect host fitness among sympatric and interacting host species. We examined whether the impact of shared parasites varied among host species within the same community. Specifically, we looked at the impacts of the acanthocephalan Acanthocephalus galaxii, the trematodes Coitocaecum parvum and Maritrema poulini, and the nematode Hedruris spinigera, on three host species: the amphipods, Paracalliope fluviatilis and Paracorophium excavatum, and the isopod, Austridotea annectens. We assessed parasite infection levels in the three host species and tested for effects on host survival, behavior, probability of pairing, and fecundity. Maritrema poulini and C. parvum were most abundant in P. excavatum but had no effect on its survival, whereas they negatively affected the survival of P. fluviatilis, the other amphipod. Female amphipods carrying young had higher M. poulini and C. parvum abundance than those without, yet the number of young carried was not linked to parasite abundance. Behavior of the isopod A. annectens was affected by M. poulini infection; more heavily infected individuals were more active. Paracorophium excavatum moved longer distances when abundance of C. parvum was lower, yet no relationship existed with respect to infection by both M. poulini and C. parvum. The differential effects of parasites on amphipods and isopods may lead to community‐wide effects. Understanding the consequences of parasitic infection and differences among host species is key to gaining greater insight into the role of parasite mediation in ecosystem dynamics.     

Pseudomonas fluorescens and Erwinia herbicola Reduce Wilt of Grasses Caused by Xanthomonas campestris pv. graminis

Pseudomonas fluorescens and Erwinia herbicola were isolated from Italian ryegrass surviving to infection by Xanthomonas campestris pv. graminis. They reduced symptom expression and mortality of young ryegrass plants when inoculated simultaneously with or prior to the parasite. Protection from wilt was a quantitative effect, depending on the ratio pathogenic/non-pathogenic bacteria: it was good with 1/99, less with 10/90 and moderate to low with 50/50. The effect of preinoculation by leaf-clipping with scissors contaminated by P. fluorescens or E. herbicola could be detected at least seven days after clipping and at a distance of 2 cm below the clipping level. Preinoculation of roots, or stem bases did not protect the plants from leaf infection by the parasite.     

Protection of Oilseed Rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) Toward Fungal Pathogens by Strains of Plant-associated <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis>

In this report, four Bacillus strains were tested for effects on plant fitness and disease protection of oilseed rape (Brassica napus). The strains belonged to newly discovered plant-associated Bacillus amyloliquefaciens and a recently proposed species, Bacillus endophyticus. The fungal pathogens tested represented different infection strategies and included Alternaria brassicae, Botrytis cinerea, Leptosphaeria maculans, and Verticillium longisporum. The B. amyloliquefaciens strains showed no or a weak plant growth promoting activity, whereas the B. endophyticus strain had negative effects on the plant as revealed by phenological analysis. On the other hand, two of the B. amyloliquefaciens strains conferred protection of oilseed rape toward all pathogens tested. In vitro experiments studying the effects of Bacillus exudates on fungal growth showed clear growth inhibition in several but not all cases. The protective effects of Bacillus can therefore, at least in part, be explained by production of antibiotic substances, but other mechanisms must also be involved probably as a result of intricate plant–bacteria interaction. The protective effects observed for certain Bacillus strains make them highly interesting for further studies as biocontrol agents in Brassica cultivation.     

RAPD-Based Inter- and Intravarietal Classification of Fungi of the gaeumannomyces-Phialophora Complex

The suitability of randomly amplified polymorphic DNAs (RAPD) for differentiation at the varietal and intravarietal level was tested on several hundred isolates of the gaeumannomyces-Phialophora (G-P) complex from different geographic locations and host plants. Amplification products obtained using two decamer primers allowed differentiation between gaeumannomyces graminis and gaeumannomyces cylindrosporus and between the three varieties of gaeumannomyces graminis, as well as further division at the intravarietal level. Thus, isolates of the causal agent of take-all on cereals, Gaeumannomyces graminis var. tritici were divided into six subgroups by amplification with these two primers. There is some evidence for an association between host preference and RAPD subgroups but further work is needed to confirm this and to determine the importance of these subgroups. This fast and easy method is a useful tool for investigating the occurrence and distribution of this pathogen and for studying changes in the populations of species and subgroups of Gaeumannomyces in cereal cropping systems     

The effect of non-pathogenic phylloplane fungi on life-history traits of urticae (Acari: Tetranychidae)

The direct effect of three common non-pathogenic phylloplane fungi on the life-history traits of Tetranychus urticae Koch was investigated on intact leaves of the garden bean Phaseolus vulgaris L. (var. Tendergreen Improved), under conditions of low and high water-deficit stress. The survival rate of T. urticae was always reduced by an increase in water-deficit stress, whereas the effect of the fungi depended in part on the watering regime. On two of the three fungi tested, Alternaria alternata (Fr.) Keissler and Epicoccum nigrum Link, the mites showed a significant increase in net reproduction compared to those reared under control (i.e. no fungi added) conditions, independent of water-deficit stress, resulting in a higher intrinsic rate of increase. The third fungus tested, Cladosporium cladosporioides (Fres.) de Vries, had a similar response with respect to net reproduction when reared under low water-deficit stress but not when reared under a high water-deficit stress. There was, however, no evidence of an interactive effect with water-deficit stress and presence or absence of fungus affecting the net reproduction of the mites, for any of the three fungal species tested. An interactive effect between fungal species and water-deficit stress was observed for the intrinsic rate of increase of the mites. It is suspected that the fungi are acting as a source of an otherwise limiting resource and the differences observed between the fungal treatments is due to differences in consumption, based on the different sizes of their conidia.     

A comparative study of Phialophora radicicola, an avirulent fungal root parasite of grasses and cereals

The biology and infection-behaviour of a typical isolate of Phialophora radicicola Cain have been compared with those of a representative isolate of Ophiobolus graminis (Sacc.) Sacc. Both species can utilize a nitrate source of nitrogen and both require thiamine and biotin for growth on inorganic nitro-gen; P. radicicola, but not O. graminis, was able to synthesize biotin when grown on asparagine as a nitrogen source. The pH range for good growth of P. radicicola in nutrient solution was narrower than that for O. graminis, and its growth rate on agar was only one-third. P. radicicola was the more active decomposer of cellulose, and its cellulolysis adequacy index was I.66 as com-pared with a value of 0.33 for 0. graminis. In agreement with prediction from Garrett's (I966) hypothesis on the cellulolysis adequacy index, saprophytic survival of P. radicicola in wheat straw was shortened by additional soil nitrogen, which prolongs survival of O. graminis.P. radicicola was found to spread ectotrophically over the roots of wheat, oats and barley by runner hyphae indistinguishable from those of O. graminis, but cortical infection caused no necrosis and no discernible check to growth of the infected cereals, nor any significant decrease in grain yield of inoculated wheat grown to maturity. Pre-existing infection of wheat roots by P. radicicola retarded spread of infection by O. graminis; inoculation of several grass species with P. radicicola reduced the extent of infection by O. graminis of wheat following the grasses.     

Factors affecting the adsorption of buchnericin LB, a bacteriocin produced by Lactocobacillus buchneri

Buchnericin-LB adsorbs to gram-positive but not to gram-negative bacteria. The tested gram-positive bacteria were species of Lactobacillus, Pediococcus, Leuconostoc, Enterococcus, Lactococcus, Listeria, Bacillus, Staphylococcus; gram-negative bacteria belonged to the genera Salmonella, Escherichia, Yersinia and Pseudomonas. Buchnericin-LB adsorption depended on pH but not on time and temperature. Also some anions of salts and lipoteichoic acid reduced or inhibited its adsorption. Treatment of cells and cell walls of sensitive bacteria with detergents, organic solvents or enzymes did not affect subsequent binding of buchnericin-LB. Treatment with buchnericin-LB caused sensitive cells to lose high amounts of intracellular K⁺ ions and UV-absorbing materials and became more permeable to o-nitrophenol-β-D-galactopyranoside. Buchnericin-LB (640-2560 AU/ml) decreased the colony forming units (99%) and absorbance values of Listeria monocytogenes and Bacillus cereus. These results indicate that the mode of action of buchnericin-LB is bactericidal and its lethal effect is very rapid.     

Some Physiological Aspects of Bacterial Wilt of Forage Grasses Caused by Xanthomonas campestris pv. graminis

The 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.