Analysis of a Secreted Aspartic Peptidase Disruption Mutant of Glomerella cingulata

Peptidases have been implicated in the pathogenicity of fungi that cause disease in plants. Expression of the secreted aspartic peptidase gene (gcsap), of a Glomerella cingulata isolate pathogenic on apples, is induced during appressorium formation. To determine whether the secreted aspartic peptidase (GcSAP) is essential to pathogenicity, gcsap was disrupted using a vector containing a 637 bp fragment of genomic DNA that encodes the sequence spanning the two active site aspartic acid (Asp) residues. To ensure that the truncated gcsap gene products could not have residual peptidase activity the codons for the active site residues Asp¹¹² and Asp²⁹⁷ were both mutated to histidine residues. Both PCR and Southern analysis confirmed disruption of gcsap. Neither gcsap mRNA nor GcSAP activity was detected in the disruption mutant. Pathogenicity tests on fruit from three apple cultivars showed that GcSAP was not required for pathogenicity. The disruption mutant grew on medium containing protein as the sole source of nitrogen because G. cingulata secretes a previously undetected peptidase(s). A serine peptidase that had a pH optimum between pH 7.0 and 8.0 and a K _m of 0.25 mM for the synthetic substrate succinyl-Ala–Ala–Pro–Phe-p-nitroanilide was identified.     

PGPR and entomopathogenic fungus bioformulation for the synchronous management of leaffolder pest and sheath blight disease of rice

BACKGROUND: The biological control of plant pests and diseases using a single organism has been reported to give inconsistent and poor performance. To improve the efficacy, bioformulations were developed possessing mixtures of bioagents. RESULTS: Bioformulations combining Pseudomonas fluorescens Migula strains Pf1 and AH1 and Beauveria bassiana (Balsamo) Vuill. isolate B2 were developed and tested for their efficacy against leaffolder pest and sheath blight disease on rice under glasshouse and field conditions. The combination of Pf1, AH1 and B2 effectively reduced the incidence of leaffolder insect and sheath blight disease on rice compared with other treatments. An in vitro assay of leaffolder preference to rice leaf tissues treated with Pf1 + AH1 + B2 biformulation showed variation from normal growth and development of leaffolder larvae. Plants treated with the Pf1 + AH1 + B2 combination showed a greater accumulation of enzymes, lipoxygenase and chitinase activity against leaffolder insect compared with other treatments. Similarly, the plants showed a higher accumulation of defence enzymes, peroxidase and polyphenol oxidase activity against sheath blight pathogen in Pf1 + AH1 + B2 treatment compared with the untreated control. The bioformulation mixture attracted the natural enemy population of leaffolder under field conditions. In addition, a significant increase in rice grain yield was observed in Pf1 + AH1 + B2 treatment compared with the untreated control. CONCLUSION: The combination of P. fluorescens strains and B. bassiana isolate effectively reduced the incidence of leaffolder insect and sheath blight disease on rice plants and showed the possibility of controlling both pest and disease using a single bioformulation. Copyright © 2010 Society of Chemical Industry     

Competitiveness of native Bradyrhizobium japonicum strains in two different soil types

Interstrain competitiveness is a key factor affecting the performance of rhizobium inoculant. In the present study five native strains of Bradyrhizobium japonicum, namely SSF 4, SSF 5, SSF 6, SSF 7 and SSF 8, were assessed for their competitiveness in nodulating soybean using serological methods. The strains were inoculated individually or with the type strain USDA 110 at a 1:1 ratio. Nodule occupancy determined by immunofluorescence and dot immunoblot assay revealed that under in vitro conditions SSF 8 is more competitive than USDA 110 whereas the others were less competitive. The competitive ability of these strains was also estimated in pot culture in the field. In red soil both SSF 8 and USDA 110 were equally competitive whereas in black soil SSF 8 competed better than USDA 110 and produced more nodules. In a black soil field trial using a randomized block design, USDA 110 or SSF 8, when inoculated alone, occupied the majority of the nodules and enhanced nodule dry weight and shoot biomass. SSF 8 was more competitive when the strains were co-inoculated. Received: 1 November 1996     

Total and soluble fluorine concentrations in relation to properties of soils in New Zealand

Soil fluorine (F) concentrations continue to increase in agricultural soils receiving regular applications of phosphatic fertilizer. Continued accumulation of soil F poses a risk to grazing ruminants and may pose a future risk to groundwater quality. This paper examines the range of total F (F_t) concentrations and forms of soluble F species and their relationship to selected soil properties in New Zealand agricultural soils. The F_t and soluble F (soil F extracted with water (F_water) and 0.01 m KCl (F_KCl)) concentrations in 27 soil samples (0–75 mm depth) taken from predominantly pasture sites in the North and South Islands of New Zealand were much less than those reported in the literature for sites contaminated with F from industry. The F_t concentrations ranged from 212 to 617 µg F g^?1 soil. The F‐toxicity risk to grazing animals in farms at these sites through soil ingestion is small at present, but farms with very large F_t concentrations (i.e. > 500 µg F g^?1) need to adopt suitable grazing and fertilizer management practices to avoid future F‐toxicity risk. The F_t concentration had very strong positive correlations with both total soil P and total soil Cd concentrations, reflecting the link between P fertilizer use and F accumulation in the soils. It also had significant positive correlations with organic matter and amorphous Al oxides contents, indicating that F is strongly bound to Al polymers adsorbed to organic matter and amorphous Al oxides. The F_water and F_KCl concentrations and free F^– ion concentrations in water (F^–_water) and 0.01 m KCl (F^–_KCl) extracts were generally two and three orders of magnitude, respectively, less than the F_t concentrations and were much less than the concentrations considered phytotoxic. The F_water and F_KCl concentrations were positively related to soil organic matter content and negatively related to soil pH. Regression models relating F_water and F_KCl concentrations to soil organic matter content and soil pH suggest that F can be very soluble in extremely acidic soils (pH(water) < 4.9) with large organic matter contents and therefore F potentially may contaminate groundwater if these soils are also coarse‐textured and the water table is shallow.     

Tree species as hosts for arbuscular mycorrhizal and dark septate endophyte fungi

A survey of 35 tree species(belonging to 28 genera in 19 families) in Aliyar,South India was carried out to ascertain their arbuscular mycorrhizal(AM) and dark septate endophyte(DSE) fungal status.All the tree species examined had AM association.AM and DSE colonization is reported for the first time in 20 and 14 species respectively.Cooccurrence of AM and DSE was observed in 14(40%) tree species.The extent of DSE colonization was inversely related to the extent of AM fungal colonization.Six tree species had Arum-type,18 had intermediatetype and 11 had typical Paris-type AM morphology.AM fungal spore morphotypes belonging to 11 species in two genera were isolated from the rhizosphere soil.AM fungal spore numbers were not related to the extent of AM colonization and Glomus dominated spore diversity.AM association individually and along with DSE were found respectively in the 63% and 44% of the economically important tree species.The occurrence of AM and DSE fungal association in economically important indigenous tree species indicates the possibility of exploiting this association in future conservation programmes of these species.     

Localized Enrichment of PCB Levels in Street Dust Due to Redistribution by Wind

As part of a project to identify PCB sources in combined sewer overflows discharging to the Buffalo River, NY, nine street and two unpaved aggregate parking lot dust and dirt samples were collected and analyzed for selected PCB congeners and total PCBs. Without exception, the dust and dirt samples had greater concentrations associated with the higher-chlorinated homologue groups. PCB-congeners 153, 138, 101, 118, and 180 contributed >50% of the total PCBs mass in each sample, suggesting contamination with Aroclors 1254 and 1260. Total PCB concentrations for the dust and dirt samples ranged between 90 and 1700 ng g^-1. Samples with the highest concentrations were located across the street and downwind from an industrial lot that had been remediated for PCB contamination in 1988. The industrial lot had been contaminated with Aroclors 1254 and 1260, indicating that contaminated soil had been locally redistributed through natural and vehicle-generated wind. Calculations based on the particle size of the street dust confirm the possibility of wind-induced redistribution. Street and aggregate lot dust and dirt potentially contribute to Buffalo River PCBs loadings through combined sewer overflows and stormwater runoff. Best management practices such as more frequent street sweeping may be useful in reducing these loadings.     

Influence of Ectomycorrhizal Hyphae on Phosphate Fractions and Dissolution of Phosphate Rock in Rhizosphere Soils of Pinus radiata

A study was conducted to determine the effect of ectomycorrhizal hyphae on phosphate rock dissolution and soil properties in the rhizosphere of one-year-old Pinus radiata D. Don seedlings established on an allophanic soil (Andosol) in the field. High and low ectomycorrhizal hyphal activities were produced in soils inside the lower compartments of rhizosphere study containers by using either 26 μm or < 10 μ m pore-size nylon mesh(es) at the interfaces between the lower and upper compartments. The meshes at the interfaces allowed (26 μm) or reduced (< 10 μ m) hyphae penetration into the lower compartment. Pairs of containers, one with 26 μ m mesh and the other with < 10 μ m mesh, were buried under Pinus radiata seedlings for 10 months in a forest plantation. The effect of ectomycorrhizal (ECM) hyphal activity on phosphate rock dissolution and soil properties was determined. The increased ECM hyphal activity induced increased soil acidification and promoted greater production of acid phosphatase enzyme, causing greater changes in phosphorus (P) fractions in the rhizosphere soils. The increased ECM hyphal activity also stimulated higher dissolution of phosphate rock and mobilization of soil organic P. The results suggest that ECM hyphal strand has a significant role in mobilizing low-solubility P forms and soil organic P for utilization by the tree.     

The effect of age on the fluoride concentration in the metacarpus of grazing sheep in New Zealand

AIM: To determine the concentration of fluoride (F) in the metacarpi from ewes of varying age, which had been grazing pastures that had been regularly topdressed with phosphatic fertilisers, which contain F as a contaminant, for at least 30 years. METHODS: Three groups of 10 ewes aged 6-8, 18-20 and 60-72 months were selected from seven research farms with a known fertiliser history, representing the major sheep farming regions of New Zealand. Lambs were born and remained on the property. The sheep were collected and slaughtered in autumn, and the right metacarpus removed for determination of F content. Representative samples of topsoil (0-30- and 30-60-mm depths) were also collected for determination of F content. RESULTS: The total concentration of F in topsoil on the farms ranged from 161 to 712 mg/kg. The overall mean concentrations of F in metacarpi from 6-8-, 18-20- and 60-72-monthold ewes were 170 (SE 9.9), 308 (SE 13.9) and 480 (SE 28.4) mg/kg dry matter (DM), respectively. On six farms, lambs which had been exposed to limited or no ingestion of soil had the lowest concentration of F in their metacarpi. Generally, there was a curvilinear relationship between age and concentration of F in the metacarpi of sheep. On the seventh farm, the concentrations of F in the metacarpi from the 6-8- and 18-20-month-old sheep were similar but lower than those of the 60-72-month-old ewes. The area of soils on the farm where the 6-8-month-old-sheep were grazing had higher total concentrations of F in topsoil (271 mg/kg) than those soils grazed by the 18-20-month-old sheep (205 mg/kg), and this may be one reason for the elevated concentration of F in the metacarpi of the younger ewes. The concentrations of F in metacarpi, at any age, were not related to the concentration of F in topsoils. CONCLUSIONS: Many factors can influence the amounts of F in soil ingested by grazing sheep and the concentration of F in their bone. The concentration of F in the metacarpi of ewes generally increased curvilinearly with age, and the rate of accumulation of F in bone was greatest in young sheep. The potential risk of chronic fluorosis occurring in sheep grazing pastures in New Zealand is low for animals managed in a manner similar to the flocks studied, given the highest mean concentration of F was 601 mg/kg DM, observed in metacarpi from 60-72-month-old ewes, and that a concentration of at least 2,400 mg/kg DM is the threshold for chronic fluorosis.     

The effect of a temporal change in ingestion rates of fluorine (F) in soil on the concentration of F in serum and bone of young sheep

AIM: To determine the changes in concentrations of fluorine (F) in serum and bone of young sheep given a high intake of F, as F in soil, followed by a low intake of F, to simulate the varying intakes experienced by grazing sheep. METHODS: Sixty autumn-born, weaned, mixed-sex lambs were randomly divided into two groups (n=30). One group (Control) was fed a low-F (12 mg/kg dry matter; DM) lucerne/ grain diet for 345 days, while the other group (High soil-F) was fed a high-F (224 mg/kg DM) lucerne/grain diet containing 10% soil for 94 days and then a low-F diet for a further 251 days. Blood samples were collected on Days 1, 32, 94, 108, 153, 214, 280 and 345, to determine concentrations of F in serum. Groups of five or six animals from each group were killed on Days 1, 94, 153, 214, 280 and 345, to collect radii and humeri for determination of F content. The sheep were weighed at about 6-8-weekly intervals. RESULTS: Initial liveweight of the sheep was 22.1 (standard error (SE) 1.12) kg and the mean liveweight gains were 270 (SE 22.1) and 170 (SE 12.4) g/day from Days 1 to 94 and 95 to 330, respectively. The mean initial (Day 1) concentration of F in serum was 0.03 (SE 0.005) mg/L, and this changed very little for animals on the low-F diet. In sheep on the high soil-F diet, the concentration of F in serum reached 0.38 (SE 0.021) mg/L at Day 94 but after being placed on the low-F diet concentrations decreased to 0.04 (SE 0.006) mg/L after 14 days (Day 108) and then remained at about this low level for the remainder of the duration of the study. The mean concentrations of F in the humerus and radius of the lambs at Day 1 were 161 (SE 19.4) and 159 (SE 20.4) mg/kg DM, respectively, which increased to 2,784 (SE 80.6) and 1,805 (SE 51.5) mg/kg DM, respectively, at Day 94 in lambs fed the high soil-F diet, then decreased to 1,075 (SE 58.1) and 1,064 (SE 61.6) mg/kg DM at Day 153, and then progressively increased to 1,669 (SE 57.7) and 1,312 (SE 30.8) mg/kg DM at Day 345. CONCLUSIONS: The ingestion of F in soil by sheep markedly increased their concentrations of F in serum and bone, but when the soil was removed and they were fed a low-F diet, concentrations of F in serum decreased to baseline levels within 14 days while concentrations in the bone only decreased to 60-70% of the peak concentration, indicating that some of the F in bone was remobilised.     

Genetic diversity and relationship between Bradyrhizobium strains isolated from blackgram and cowpea

The genetic diversity of bradyrhizobial strains associated with blackgram and cowpea grown in two different agricultural soils (non-saline and saline) along the coastline of Tamil Nadu has been analysed. Phenotypically indistinguishable isolates were analysed for DNA polymorphism using random amplification of polymorphic DNA (RAPD) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of 16S rDNA and nifD. Although these bacteria belong to a group with a broad host range, RAPD analysis showed a considerable level of genetic diversity among the strains isolated from different host plants. Soil pH and salinity seem to have an effect on the selection of natural populations as revealed by PCR-RFLP of 16S rDNA. A combination of PCR-RFLP genotyping with nodulation studies indicates that monocropping of blackgram and the salinity of the soil have made ineffective rhizobia the dominant genotype, thereby creating an ecological burden on their other compatible hosts. A group of strains and a type strain sharing three different 16S PCR-RFLP types were shown to have the same set of symbiotic genes as inferred from the PCR-RFLP pattern of nifD. Another group of cowpea rhizobia that were found to be effective nitrogen fixers and sharing distinct 16S profiles were found to have a different set of symbiotic genes.