Biological control of take-all by fluorescent Pseudomonas spp. from Chinese wheat fields

Take-all disease of wheat caused by the soilborne fungus Gaeumannomyces graminis var. tritici is one of the most important root diseases of wheat worldwide. Bacteria were isolated from winter wheat from irrigated and rainfed fields in Hebei and Jiangsu provinces in China, respectively. Samples from rhizosphere soil, roots, stems, and leaves were plated onto King's medium B agar and 553 isolates were selected. On the basis of in vitro tests, 105 isolates (19% of the total) inhibited G. graminis var. tritici and all were identified as Pseudomonas spp. by amplified ribosomal DNA restriction analysis. Based on biocontrol assays, 13 strains were selected for further analysis. All of them aggressively colonized the rhizosphere of wheat and suppressed take-all. Of the 13 strains, 3 (HC9-07, HC13-07, and JC14-07, all stem endophytes) had genes for the biosynthesis of phenazine-1-carboxylic acid (PCA) but none had genes for the production of 2,4-diacetylphloroglucinol, pyoluteorin, or pyrrolnitrin. High-pressure liquid chromatography (HPLC) analysis of 2-day-old cultures confirmed that HC9-07, HC13-07, and JC14-07 produced PCA but no other phenazines were detected. HPLC quantitative time-of-flight 2 mass-spectrometry analysis of extracts from roots of spring wheat colonized by HC9-07, HC13-07, or Pseudomonas fluorescens 2-79 demonstrated that all three strains produced PCA in the rhizosphere. Loss of PCA production by strain HC9-07 resulted in a loss of biocontrol activity. Analysis of DNA sequences within the key phenazine biosynthesis gene phzF and of 16S rDNA indicated that strains HC9-07, HC13-07, and JC14-07 were similar to the well-described PCA producer P. fluorescens 2-79. This is the first report of 2-79-like bacteria being isolated from Asia.     

Gene amplification and insecticide resistance

Pesticide resistance in arthropods has been shown to evolve by two main mechanisms, the enhanced production of metabolic enzymes, which bind to and/or detoxify the pesticide, and mutation of the target protein, which makes it less sensitive to the pesticide. One route that leads to enhanced metabolism is the duplication or amplification of the structural gene(s) encoding the detoxifying enzyme, and this has now been described for the three main families (esterases, glutathione S-transferases and cytochrome P450 monooxygenases) implicated in resistance. More recently, a direct or indirect role for gene duplication or amplification has been described for target-site resistance in several arthropod species. This mini-review summarises the involvement of gene duplication/amplification in the insecticide/acaricide resistance of insect and mite pests and highlights recent developments in this area in relation to P450-mediated and target-site resistance.     

Biological control of Rhizoctonia root rot on bean by phenazine- and cyclic lipopeptide-producing Pseudomonas CMR12a

Pseudomonas CMR12a was previously selected as an efficient biocontrol strain producing phenazines and cyclic lipopeptides (CLPs). In this study, biocontrol capacity of Pseudomonas CMR12a against Rhizoctonia root rot of bean and the involvement of phenazines and CLPs in this ability were tested. Two different anastomosis groups (AGs) of Rhizoctonia solani, the intermediately aggressive AG 2-2 and the highly aggressive AG 4 HGI, were included in growth-chamber experiments with bean plants. The wild-type strain CMR12a dramatically reduced disease severity caused by both R. solani AGs. A CLP-deficient and a phenazine-deficient mutant of CMR12a still protected bean plants, albeit to a lesser extent compared with the wild type. Two mutants deficient in both phenazine and CLP production completely lost their biocontrol activity. Disease-suppressive capacity of CMR12a decreased after washing bacteria before application to soil and thereby removing metabolites produced during growth on plate. In addition, microscopic observations revealed pronounced branching of hyphal tips of both R. solani AGs in the presence of CMR12a. More branched and denser mycelium was also observed for the phenazine-deficient mutant; however, neither the CLP-deficient mutant nor the mutants deficient in both CLPs and phenazines influenced hyphal growth. Together, results demonstrate the involvement of phenazines and CLPs during Pseudomonas CMR12a-mediated biocontrol of Rhizoctonia root rot of bean.     

Analysis of fenbendazole residues in bovine milk by ELISA

Fenbendazole residues in bovine milk were analyzed by ELISAs using two monoclonal antibodies. One monoclonal antibody (MAb 587) bound the major benzimidazole anthelmintic drugs, including fenbendazole, oxfendazole, and fenbendazole sulfone. The other (MAb 591) was more specific for fenbendazole, with 13% cross-reactivity with the sulfone and no significant binding to the sulfoxide metabolite. The limit of detection of the ELISA method in the milk matrix was 7 ppb for MAb 587 and 3 ppb for MAb 591. Fenbendazole was administered in feed, drench, and paste form to three groups of dairy cattle. Milk was collected immediately before dosing and then every 12 h for 5 days. The ELISA indicated that residue levels varied widely among individual cows in each group. Fenbendazole levels peaked at approximately 12-24 h and declined rapidly thereafter. Metabolites were detected at much higher levels than the parent compound, peaked at approximately 24-36 h, and declined gradually. Residue levels were undetectable by 72 h. The ELISA data correlated well with the total residues determined by chromatographic analysis, but the use of the two separate ELISAs did not afford an advantage over ELISA with the single, broadly reactive MAb 587. The ELISA method could be used to flag high-residue samples in on-site monitoring of fenbendazole in milk and is a potential tool for studying drug pharmacokinetics.     

Location of roosts of the lesser long-eared bat Nyctophilus geoffroyi and Gould's wattled bat Chalinolobus gouldii in a fragmented landscape in south-eastern Australia

The location of roosts of two species of vespertilionid bats, the lesser long-eared bat (Nyctophilus geoffroyi) and Gould’s wattled bat (Chalinolobus gouldii), was investigated in a remnant vegetation-farmland mosaic and adjacent floodplain forest in south-eastern Australia. A total of 45 individuals of N. geoffroyi and 27 C. gouldii were fitted with radio transmitters, which resulted in the location of 139 and 89 roosts respectively. Selection of roosting habitat showed both inter- and intra-specific differences. At the landscape level, locations of roosts used by male and female N. geoffroyi differed markedly. Most males roosted in the farmland mosaic within 3 km (mean 1.9±2.9 km) of where they were captured while foraging in remnant vegetation. In contrast, roost sites of females were predominantly in the floodplain forest, 6-12 km from their capture site in farmland (mean for all females, 6.7±2.9 km). All maternity roosts were in the extensive floodplain forest, 4-10 km from foraging areas. Distances moved by this species were greater than previously recorded and further than predicted by flight morphology. Most C. gouldii roosted in the floodplain forest, 4-10 km from their capture site (6.9±1.6 km). Within the floodplain forest, both species roosted in areas that had higher densities of hollow-bearing trees than generally available; dead hollow-bearing trees for N. geoffroyi and large, live trees for C. gouldii. The scale of movements undertaken by these species means that they can obtain resources from multiple landscape elements. Both species used different habitats for roosting and foraging despite the energetic costs of commuting relatively large distances. Conservation of bat populations in rural environments needs to be considered at the landscape scale, with particular attention to identifying landscape elements that provide key resources.     

A retrospective study of 11 cases of lungworm (Didelphostrongylus hayesi) infection in opossums (Didelphis virginiana).

A juvenile, female North American opossum (Didelphis virginiana) died of verminous pneumonia caused by Didelphostrongylus haysei despite aggressive treatment with oral fenbendazole, corticosteroids, and antibiotics. This prompted a retrospective study of lungworm infection in opossums, during which 19 additional necropsy reports from opossums were reviewed. Including the subject of this report, a total of 11 (55%) of these cases included a diagnosis of lungworm infection. This diagnosis was considered to have contributed to death in eight out of the 11 cases (73%). Histologically, 10 of the 11 (91%) opossums had granulomatous bronchopneumonia with small to moderate numbers of adult nematodes in the airways and parenchyma. Four of the 11 (36%) opossums had free larvae within the parenchyma or terminal airways. Inflammation was usually associated with larvae, degenerating parasites, and nonintact adult nematodes. Superimposed bacterial pneumonia was evident in three animals, and sections of lung examined from all the opossums were characterized by moderate to severe smooth-muscle hyperplasia in airways, including terminal respiratory bronchioles and alveolar ducts. Nine animals had prominent medial smooth-muscle hyperplasia in small- and medium-sized arterioles. Lesions in other organs, particularly in liver, heart, and gastrointestinal tract, were frequently identified. Three animals had concomitant septicemia or bacterial bronchopneumonia (or both), which contributed to the cause of death. Seven animals had gastric nematodosis (Physaloptera sp.), although three of them had been treated with a 14-day course of fenbendazole.     

Blastomycosis in nondomestic felids.

Blastomycosis was diagnosed in six nondomestic felids from eastern Tennessee, including two Asian lions (Panthera leo persicus), one African lion (Panthera leo), one Siberian tiger (Panthera tigris), one cheetah (Acinonyx jubatus), and one snow leopard (Panthera uncia). Clinical signs included lethargy, anorexia, weight loss, dyspnea, sneezing. ataxia, and paresis. Variable nonspecific changes included leukocytosis, monocytosis, moderate left shift of neutrophils, moderate hypercalcemia, hyperproteinemia, and hyperglobulinemia. Thoracic radiographs revealed interstitial and alveolar changes, consolidation or collapse of a lung lobe, bullae formation, and a pulmonary mass. Agar gel immunodiffusion (AGID) serology for Blastomyces dermatitidis was performed in five felids and was positive in three. The tiger had cerebral blastomycosis and was positive for AGID serologic tests of both cerebrospinal fluid and serum. One percutaneous lung aspirate in the snow leopard and one bronchial aspirate in an Asian lion demonstrated B. dermatitidis organisms. whereas tracheal wash samples and a nasal discharge were nondiagnostic in others. Treatment with itraconazole was attempted in four cats. The tiger improved before euthanasia, whereas the others did not survive beyond initial treatments. In four felids, B. dermatitidis was found in the lungs and tracheobronchial lymph nodes associated with a florid pyogranulomatous reaction; the tiger had a pyogranulomatous encephalomyelitis, and the cheetah had a single pulmonary granuloma. Thoracic radiography, cytologic examination of lung lesion aspirates, and B. dermatitidis AGID serology should be performed on clinically ill zoo felids in endemic areas to rule out blastomycosis.