Microbial pathogens of the coconut pest Oryctes rhinoceros: influence of weather factors on their infectivity and study of their coincidental ecology in Kerala,India

The rhinoceros beetle, Oryctes rhinoceros L., is an economically important pest of the coconut palm. Management of this pest has been accomplished using microbial agents viz., Oryctes virus (OrV) and an entomofungal pathogen Metarhizium anisopliae. Recently an opportunistic bacterial pathogen Pseudomonas alcaligenes has also been noticed to cause septicaemia in the grubs when under stress. To unravel the influence of abiotic weather factors and the interactions amongst these microbial pathogens, a 3 year study was conducted from September 1996 to August 1999 in three of the southern districts of Kerala, India. Of the 6627 grubs and 307 adults collected from various breeding sites of the pest, 5% of the grubs and 22% of the adults had natural virus infection, 3% larvae died of M. anisopliae mycosis and 20% larvae succumbed to bacterial septicaemia. Oryctes virus infection in grubs and adults was negatively correlated to minimum temperature (correlation co-efficient, r = –0.4, and –0.6 respectively, sample size, n = 0). Increase in relative humidity increased the fungal activity (r = 0.8) whereas, maximum temperature had a negative impact (r = –0.7). Occurrence of virus infection in grubs and adults was positively correlated (r = 0.6), supporting the contention of active transmission of the virus pathogen between these two stages. The bacterial septicaemia in the grubs was marginally correlated with virus infection and P. alcaligenes undermined the efficiency of the virus pathogen.     

Analysis of esterase zymograms ofFusarium sambucinum and related species

Esterase zymograms were obtained following polyacrylamide slab gel electrophoresis of protein extractsFusarium sambucinum and related species originating from different geographic locations and different matrices. The sites of esterase activity were recorded, and the R_fs were calculated. The data were used for the construction of phenograms by cluster analysis and nonlinear mapping by computerized classification techniques. The fifteen isolates ofF. sambucinum, the eight isolates ofF. torulosum and the six isolates ofF. spec. nov. each had identical profiles, and are therefore electrophoretically distinct species. The isolates ofF. sarcochroum, one ofF. sambucinum sensu lato (BBA 64280) and fifteen isolates ofF. sambucinum were electrophoretically indistinguishable from each other. We assume they are synonymous. The isolate ofF. bactridioides, one ofF. sambucinum sensu lato (BBA 64993) and eight isolates ofF. torulosum had uniform EST patterns, therefore the two species are electrophoretically identical. We assume they are also synonymous. The remaining three isolates ofF. sambucinum sensu lato are somewhat closely related toF. sambucinum isolates on the basis of our investigations.     

Characterization and infectivity of a spontaneous variant isolated fromFrankia sp. WEY 0131391

Summary A spontaneous variant, obtained from aFrankia isolate fromAlnus rubra nodules, was compared with the parent strain with regard to infectivity, nitrogenase activity, and electrophoretic and immunological profiles. Both the parent and the variant strain were equally effective in inducing nodulation in seedlings ofA. rubra. All inoculated plants had an active nitrogenase system as measured by the acetylene reduction assay. Electrophoresis of whole cell homogenates on SDS-polyacrylamide slab gels showed similar electrophoretic profiles; however, the variant strain also exhibited striking differences in protein patterns that distinguish it from the parent strain. Immunological analysis of the originalFrankia strain and its variant revealed shared antigens as well as immunologically distinct antigenic determinants in the two strains. The variant strain exhibits a distinct morphology and growth patterns which remain stable after many passages through culture.     

Cloning of ribosomal RNA genes from an Indian isolate ofGiardia lamblia and the use of intergenic nontranscribing spacer regions in the differentiation ofGiardia from other enteric pathogens

The ribosomal RNA genes from an Indian isolate ofGiardia lamblia have been cloned and characterized with respect to size, composition and copy number. Southern blotting and rDNA cloning ofGiardia lamblia revealed that genes coding for ribosomal RNA (rRNA) are exceptionally small and are encoded within a 5.6 kb genome fragment repeat. The rDNA repeat unit of this isolate was found to be highly G-C rich like other human isolates and the physical map showed severalSmaI sites. There are 132 copies of the rDNA repeat unit per cell in a head to tail arrangement. Two fragments corresponding to intergenic (0.2 kb and 0.3 kb) region and one (0.8 kb) containing both an intergenic region and a small part of the small subunit ribosomal RNA (SS rRNA) have been identified within the rDNA. These were used in heterogeneity studies ofGiardia isolated from two geographic locations as well as in the analysis of cross reactivity with other enteric organisms. In Southern blots, all the three fragments were found to be highly specific for the differential diagnosis ofGiardia spp. from the other enteric pathogens. These findings should help in developing a sensitive and more specific method for the diagnosis of giardiasis over currently available techniques.     

Comparative HPLC analysis of seed albumins fromVicia faba andV. kalakhensis (Fabaceae)

Previously reported electrophoretic seed albumin data have shown an unexpected association ofVicia faba withV. kalakhensis. In the present work, seed albumins ofV. faba (subsp.paucijuga and subsp.faba) were compared with those ofV. kalakhensis using ionexchange (IE) and reversed-phase (RP) high-performance liquid chromatography (HPLC). Two subspecies ofV. faba displayed similar seed albumin profiles. On the other hand, seed albumin profiles ofV. faba andV. kalakhensis showed no major protein peak in common either in IE-HPLC or RP-HPLC chromatograms. The reported differences in seed albumin composition ofV. faba andV. kalakhensis are consistent with other taxonomical data showingV. faba to be genetically distant from the wild relatives.     

The Selection of an Isolate of the Hyphomycete Fungus,Metarhizium anisopliae,for Control of Termites in Australia

Ninety-three isolates ofMetarhizium anisopliae,mostly derived from a survey of termite material, were screened for activity againstNasutitermes exitiosusandCoptotermes frenchiorC. acinaciformisusing a grooming assay technique. Twenty-six of the most promising isolates were further evaluated by bioassay againstN. exitiosusandC. acinaciformis.All isolates were pathogenic withCoptotermesspp. being more susceptible thanN. exitiosus.A group of nine isolates, chosen for their level of pathogenicity for one or other genus of termites and to represent a genetically diverse group, was finally compared in a minicolony test using termite colonies in 1 liter jars. The isolate, code-named FI-610 (derived from nest-mound material ofC. lacteusin SE New South Wales), was one of the most effective isolates against termites from both of the two colonies tested. This isolate also grew relatively well on agar plates at 36°C. FI-610 was thus selected for field trials and was found to be effective in killing colonies ofC. acinaciformiswhen 10 g (=3 × 10¹¹conidia) or more of conidial powder was blown into the center of the large mound colonies.     

Morphological and cytological investigations ofUlothrix zonata and taxonomy of the related species

The present communication deals with the observations made on the morphology, reproduction and cytology ofUlothrix zonata (Weber & Mohr)Kütz. in culture. The alga displays a remarkable phenotypic plasticity in nature as well as in culture. The present study provides additional evidence of karyology in support ofLokhorst andVroman's treatment ofU. zonata which merges a number of earlier described species in it. The cytological details and chromosome number (n = 10) determined for the Indian isolates ofU. zonata agree with those ofSarma for the British material.NBRI Research Publication No. 63 (N.S.)     

Impact of clear-cutting and prescribed burning on microbial diversity and community structure in a Jack pine (Pinus banksiana Lamb.) clear-cut using Biolog Gram-negative microplates

Three isolates ofFusarium avenaceum are pathogenic on spotted knapweed(Centaurea maculosa), a major weed plant of pasturelands and rangelands of the Pacific Northwestern USA. One isolate (no. 1) obtained from the European centre of origin of knapweed and isolate no. 365 native to Montana, did not significantly affect knapweed seed germination. However,F. avenaceum no. 1003, another Montana native isolate, caused a 100% decrease in seed germination and hence, no seedling emergence. When formulated, isolate no. 1003, could be recovered from treated soils after 7 days and caused a significant reduction in seedling emergence or seedling dry weight. This organism had no effect on the germination ofTriticum aestivum orMedicago sativa, but did affect the germination of other plant species.F. avenaceum appears to be a candidate for the biocontrol of spotted knapweed, however, a native isolate is potentially more effective than an isolate obtained from the centre of origin ofC. maculosa.     

Hypervariable spacer regions are good sites for developing specific PCR-RFLP markers and PCR primers for screening actinorhizal symbionts

While the ribosomal RNA like highly conserved genes are good molecular chronometers for establishing phylogenetic relationships, they can also be useful in securing the amplification of adjoining hyper-variable regions. These regions can then be used for developing specific PCR primers or PCR-RFL profiles to be used as molecular markers. We report here the use of ITS region ofrrn operon ofFrankia for developing PCR-RFL profiles capable of discriminating between closely related frankiae. We have also made use of the ITS 1 region of the nuclearrrn operon ofAlnus nepalensis (D Don) for designing a PCR primer for specific amplification of nuclear DNA of this tree.     

Amylostereum laevigatum associated with the Japanese horntail,Urocerus japonicus

The fungus associated with the Japanese horntail,Urocerus japonicus, in Kochi, Kagawa and Ehime Prefectures was studied. Cultures isolated from the mycangia of 113 adult females of the horntail showed the same cultural characteristics. Four of basidiocarps found on felled logs ofCryptomeria japonica were identifieds asAmylostereum laevigatum based on morphological characteristics. This was the first record ofA. laevigatum from Japan. The cultures isolated from the basidiocarps had the same cultural characteristics as those from the mycangia ofU. japonicus. One mycangial isolate produced basidiocarps on artificially inoculated stem segments ofCr. japonica after a 6-mo incubation and was identified asA. laevigatum. One isolate from the basidiocarps ofA. laevigatum and one from the mycangium ofU. japonicus were artificially inoculated into five trees each ofChamaecyparis obtusa andCr. japonica. The wood of all inoculated trees showed discoloration, with no difference in shape and pattern of discoloration between the two isolates. The inoculated fungi were reisolated from the areas of discoloration in the inoculated trees.     

Morphological and physiological comparisons ofHelicobasidium mompa andH. purpureum

Morphological and physiological comparisons were made of sevenHelicobasidium mompa isolates and fourH. purpureum isolates. Colonies of theH. mompa isolates were thin, dense, or hard and dense, and most were pale brown to brown or dark brown, while that of isolate 344c was pinkish. Colonies ofH. purpureum isolates were hard and dense, and their colonies were dark brown. Diameters of hyphae were similar forH. mompa andH. purpureum. Dimensions of conidia and morphology of conidiophores ofH. mompa isolate 344c were close to those ofH. purpureum reported previously.H. mompa isolates grew well at 23°C, 25°C or 27°C, while all isolates ofH. purpureum grew well at 23°C. Growth rates ofH. purpureum isolates was almost the same as those ofH. mompa isolates with slow growth. Polygaracturonase activity at pH 3 was variable among the isolates for bothH. mompa andH. purpureum. Itaconic acid was produced abundantly by three isolates ofH. mompa but not produced by isolate AH130, whereas all isoaltes ofH. purpureum produced a small amount of itaconic acid.     

Experimental formulations ofBacillus sphaericus for the control of anopheline and culicine larvae

Summary Four experimental formulations ofBacillus sphaericus Neide (2362 isolate) were evaluated for larvicidal activity against culicine and anopheline larvae in several natural and artificial habitats. A granular formulation (5% primary powder) was tested against natural populations of mosquitoes in two simulated habitats in Florida and in maturing and reflooded rice fields in Louisiana. Larvae ofCulex quinquefasciatus Say were reduced by 97 and 99% after application of the granules at the rate of 10 kg/ha to polluted tanks and 2.5 kg/ha to sod-lined potholes, respectively. Anopheline andPsorophora columbiae (Dyar and Knab) larvae were reduced by 68 and 92–100%, respectively, after application of 5 kg granules/ha to rice fields. A flowable concentrate (12.8% primary powder) applied to unpolluted and organically enriched habitats in Florida at 0.25 kg/ha reduced populations ofCulex spp. by 93–100% and 99%, respectively. Sustained-release briquets (5% primary powder) applied at the rate of one half briquet/1.8 m² sod-lined potholes reduced larval populations ofCx. quinquefasciatus by 88–95% for up to 2 weeks in open sunlight. Sustained-release pellets (30% primary powder) applied to small woodland pools in Memphis, TN at the rate of four pellets/pool virtually eliminated larval populations ofCx. restuans Theobald for over 8 days. Variable persistance of larvicidal activity was noted for the other treatments depending on the formulation, target species and habitat.     

Flavonoid chemistry ofBlennospermatinae, a trans-Pacific disjunct subtribe ofSenecioneae (Asteraceae)

The flavonoid profiles of seven species ofAbrotanella and one species ofIschnea have been shown to be based upon kaempferol 3- and quercetin 3-O-glycosides and a delphinidin glycoside. Glucosides, glucuronides, arabinosides, diglucosides, and rutinosides of the flavonols were identified. The profile ofIschnea consisted solely of quercetin 3-O-glucoside and 3-O-arabinoside whereas the profiles of theAbrotanella species were more varied. Although infraspecific variation was not investigated in this study, the flavonoid chemistry of the two genera is in accordance with the flavonoid variation described for other members ofSenecioneae which are primarily flavonol producers. Based on the known phylogeny and biogeography, the flavonoid distribution from the perspective of long-distance dispersals across the Pacific is discussed. Such events should lead to genetic bottle-neck situations and depauperate flavonoid profiles. A summary of current flavonoid knowledge in theSenecioneae is supplied.     

Electrophoretic protein patterns of three species ofPhytophthora associated with black pod disease of cocoa (Theobroma cacao L.)

When electrophoretic profiles of native proteins from vegetative mycelia ofPhytophthora palmivora, Phytophthora capsici and Phytophthora citrophthora causing black pod disease of cocoa in India were compared on a single Polyacrylamide gel, the isolates of same species were readily distinguished both qualitatively by visual similarity in banding patterns and quantitatively by calculating similarity coefficients. Similarity coefficients were generally much higher between isolates within a species than between isolates of different species. The dendrograms obtained after unweighted pair grouping with arithmetic averaging cluster analysis, revealed that all the isolates ofPhytophthora capsici were highly homogenous and formed a single cluster. The isolates ofPhytophthora citrophthora were resolved into two electrophoretic types which were clustered into two distinct sub groups.Phytophthora palmivora formed a separate group. Thus, the results reveal that polyacrylamide gel electrophoresis can be used successfully in distinguishing species and sub groups within a species ofPhytophthora encountered on cocoa. CPCRl contribution No. 914.     

Phage specificity and lipopolysaccarides of stem- and root-nodulating bacteria (Azorhizobium caulinodans, Sinorhizobium spp., and Rhizobium spp.) of Sesbania spp

Phage susceptibility pattern and its correlation with lipopolysaccharide (LPS) and plasmid profiles may help in understanding the phenotypic and genotypic diversity among highly promiscuous group of rhizobia nodulating Sesbania spp.; 43 phages were from two stem-nodulating bacteria of S. rostrata and 16 phages were from root-nodulating bacteria of S. sesban, S. aegyptica and S. rostrata. Phage susceptibility pattern of 38 Sesbania nodulating bacteria was correlated with their LPS rather than plasmid profiles. Different species of bacteria (A. caulinodans- ORS571, SRS1-3 and Sinorhizobium saheli- SRR907, SRR912) showing distinct LPS subtypes were susceptible to different group of phages. Phages could also discriminate the strains of Si. saheli (SSR312, SAR610) possessing distinct LPS subtypes. Phages of Si. meliloti (SSR302) were strain-specific. All the strains of R. huautlense having incomplete LPS (insignificant O-chain) were phage-resistant. In in vitro assay, 100% of the phages were adsorbed to LPS of indicator bacterium or its closely related strain(s) only. These observations suggest the significance of LPS in phage specificity of Sesbania nodulating rhizobia. Highly specific phages may serve as biological marker for monitoring the susceptible bacterial strains in culture collections and environment.     

Vegetative compatibility of an isolate ofVerticillium dahliae pathogenic to both tomato and pepper

An isolate ofVerticillum dahliae Vdp-4, pathogenic to both tomato and pepper (tomato-pepper pathotype), was examined for its vegetative compatibility with testers of the Japanese vegetative compatibility group (subgroups J1, J2, and J3). Seven isolates ofV. dahliae from the same field as Vdp-4 in Misato, Nagano Pref. and two isolates from Hokkaido were separately determined as either tomato pathotype (B) or pepper pathotype (C). Isolate 5922 previously reported as tomato-pepper pathotype was also examined. Compatiblenit1 and NitM mutants were obtained from all isolates except for isolates Vdp-3 and Vdt-10. The isolate of tomato-pepper pathotype Vdp-4 showed a strong reaction with VCGJ1 and J3 and was thus assigned to J3. Seven of these isolates showed compatibility and were assigned into three provisional subgroups. The isolate 5922 was self-incompatible.     

Isolation of Plasmid DNA rescued from single colonies of<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> by means of rolling circle amplification

We report a simple method to isolate plasmids from single colonies ofAgrobacterium tumefaciens by means of rolling circle amplification. The amplified DNA can be digested by restriction enzymes for plasmid verification and transformed intoEscherichia coli for plasmid rescue. Compared with conventional procedures, this method eliminates liquid culturing ofAgrobacterium cells and subsequent DNA isolation and enables large-scale plasmid analyses.     

Vocal communication of wild bonnet macaques (Macaca radiata)

Field observations and spectrographic analyses of sound recordings of South Indian bonnet macaques revealed a vocal repertoire of at least 25 basic patterns. The repertoire consists of well separated sound classes and acoustic categories connected by structural intergradation. Besides structural variations within and between different elements of the repertoire, the vocal system ofMacaca radiata is characterized by regular combinations of particular basic patterns. These combinations occurred not only between calls of similar structure and function but also between calls usually emitted in entirely different social contexts. According to the qualitative analysis, sex-specific asymmetries of the vocal behaviour were less pronounced than age-dependent characteristics. The comparison of clear call vocalizations ofMacaca radiata andM. fuscata revealed significant species-specific differences on the structural and the behavioural level. Evaluations of the structural features of alarm calls of various macaque species imply marked differences between members of thefascicularis group andsinica group on one hand and thesilenus group andarctoides group on the other.     

Comparison of four media for the isolation ofAspergillus flavus group fungi

Four agar media used to isolate aflatoxin producing fungi were compared for utility in isolating fungi in theAspergillus flavus group from agricultural soils collected in 15 fields and four states in the southern United States. The four media wereAspergillus flavus andparasiticus Agar (AFPA, 14), the rose bengal agar described by Bell and Crawford (BCRB; 3), a modified rose bengal agar (M-RB), and Czapek's-Dox Agar supplemented with the antibiotics in BC-RB (CZ-RB). M-RB was the most useful for studying the population biology of this group because it permitted both identification of the greatest number ofA. flavus group strains and growth of the fewest competing fungi. M-RB supported an average of 12% moreA. flavus group colonies than the original rose bengal medium while reducing the number of mucorales colonies and the number of total fungi by 99% and 70%, respectively. M-RB was successfully employed to isolate all three aflatoxin producing species,A. flavus, A. parasiticus andA. nomius, and both the S and L strains ofA. flavus. M-RB is a defined medium without complex nitrogen and carbon sources (e.g. peptone and yeast extract) present in BC-RB. M-RB should be useful for studies on the population biology of theA. flavus group.Abbreviations M-RB Modified Rose Bengal Agar - CZ-RB Czapeks Rose Bengal Agar - BC-RB Bell and Crawford's Rose Bengal Agar - AFPA Aspergillus flavus andparasiticus agar