Field-collected Isolates Research Articles

Susceptibility of the adult house fly (Diptera: Muscidae) and 3 of its principal parasitoids (Hymenoptera: Pteromalidae) to the GHA strain of Beauveria bassiana and 4 isolates from field-collected muscid flies.

House fly (Musca domestica L.) (Diptera: Muscidae) populations can negatively impact poultry layer facilities, posing a risk to human and animal health and egg food safety. House flies quickly develop resistance to traditional chemical control methods; therefore, improved biological control may provide opportunities for improved integrated pest management (IPM) programs. Biological control methods currently used include augmentative releases of pteromalid pupal parasitoids and application of the fungal entomopathogen Beauveria bassiana (Balsamo) Vuillemin. This study used bioassays to compare the impact of different B. bassiana strains on survival of house flies and of 3 species of filth fly parasitoids. The B. bassiana that were compared were 3 new field-collected isolates, an older field-collected isolate (L90), and a common commercially available strain (GHA). Flies and parasitoids were exposed to filter paper treated with 1.5 × 109 spores of each strain and a control. All field-isolated strains induced lower mean survival times in house flies than GHA did. The results for all species of parasitoids demonstrated less difference among the treatment groups and the control than in-house flies. Although there was some effect of B. bassiana exposure on parasitoid mortality, the expected spatial separation of parasitoids from areas of application may offer some protection. Using the most effective tested strains of B. bassiana and filth fly parasitoids jointly could be a biological component of an IPM plan for fly control in poultry facilities.

Virulence Profiles and Genome-Wide Association Study for Ascochyta lentis Isolates Collected from Australian Lentil-Growing Regions.

Ascochyta lentis, the causal organism of Ascochyta blight (AB) of lentil (Lens culinaris), has been shown to produce an avirulence effector protein that mediates AB resistance in certain lentil cultivars. The two known forms of the effector protein were identified from a biparental mapping population between isolates that have reciprocal virulence on 'PBA Hurricane XT' and 'Nipper'. The effector AlAvr1-1 was described for the PBA Hurricane XT-avirulent isolate P94-24 and AlAvr1-2 characterized in the PBA Hurricane XT-virulent isolate AlKewell. Here, we performed a genome-wide association study to identify other loci associated with AB for a differential set of lentil cultivars from a diverse panel of isolates collected in the Australian lentil-growing regions from 2013 to 2020. The chromosome 3 AlAvr1 locus was strongly associated with the PBA Hurricane XT, 'Indianhead', and Nipper disease responses, but one other genomic region on chromosome 11 was also associated with the Nipper disease trait. Our results corroborate earlier work that identified the AlAvr1 locus for field-collected isolates that span the period before release and after widespread adoption of PBA Hurricane XT. A multiplex PCR assay was developed to differentiate the genes AlAvr1-1 and AlAvr1-2 to predict PBA Hurricane XT avirulence and pathotype designation in the diversity panel. Increasing numbers of the PBA Hurricane XT-virulent pathotype 2 isolates across that time indicate strong selection for isolates with the AlAvr1-2 allele. Furthermore, one other region of the A. lentis genome may contribute to the pathogen-host interaction for lentil AB.

Signatures of hybridization in Trypanosoma brucei.

Genetic exchange among disease-causing micro-organisms can generate progeny that combine different pathogenic traits. Though sexual reproduction has been described in trypanosomes, its impact on the epidemiology of Human African Trypanosomiasis (HAT) remains controversial. However, human infective and non-human infective strains of Trypanosoma brucei circulate in the same transmission cycles in HAT endemic areas in subsaharan Africa, providing the opportunity for mating during the developmental cycle in the tsetse fly vector. Here we investigated inheritance among progeny from a laboratory cross of T. brucei and then applied these insights to genomic analysis of field-collected isolates to identify signatures of past genetic exchange. Genomes of two parental and four hybrid progeny clones with a range of DNA contents were assembled and analysed by k-mer and single nucleotide polymorphism (SNP) frequencies to determine heterozygosity and chromosomal inheritance. Variant surface glycoprotein (VSG) genes and kinetoplast (mitochondrial) DNA maxi- and minicircles were extracted from each genome to examine how each of these components was inherited in the hybrid progeny. The same bioinformatic approaches were applied to an additional 37 genomes representing the diversity of T. brucei in subsaharan Africa and T. evansi. SNP analysis provided evidence of crossover events affecting all 11 pairs of megabase chromosomes and demonstrated that polyploid hybrids were formed post-meiotically and not by fusion of the parental diploid cells. VSGs and kinetoplast DNA minicircles were inherited biparentally, with approximately equal numbers from each parent, whereas maxicircles were inherited uniparentally. Extrapolation of these findings to field isolates allowed us to distinguish clonal descent from hybridization by comparing maxicircle genotype to VSG and minicircle repertoires. Discordance between maxicircle genotype and VSG and minicircle repertoires indicated inter-lineage hybridization. Significantly, some of the hybridization events we identified involved human infective and non-human infective trypanosomes circulating in the same geographic areas.

Two Novel Bacillus Strains (subtilis and simplex Species) with Promising Potential for the Biocontrol of Zymoseptoria tritici, the Causal Agent of Septoria Tritici Blotch of Wheat.

Two novel Algerian field-collected isolates were selected for their antifungal activity against Zymoseptoria tritici (teleomorph Mycosphaerella graminicola). The novel strains, termed Alg.24B1 and Alg.24B2, were identified as Bacillus subtilis and Bacillus simplex since their respective nucleotide sequences of the 16S rRNA gene were 100% and 99.93% identical to those of B. subtilis and B. simplex, respectively. The antifungal activities of Alg.24B1 and Alg.24B2 were evaluated by the well diffusion method and compared to those of other Bacillus species. The maximum activity was obtained after two days of confrontation of the bacterial strain supernatants with the fungus for Alg.24B1 and three days for Alg.24B2. Furthermore, the metabolites responsible for the antifungal activity of both strains were detected by the investigation of either gene presence (PCR) or molecule production (activity detection of lytic enzymes and HPLC detection of lipopeptides). Overall, this study showed that in addition to their ability to produce lytic enzymes (protease and β-glucanase), both strains coproduce three types of lipopeptides viz. surfactin, iturin, and fengycin. Thus, the biofungicide activity of both strains may be a result of a combination of different mechanisms. Therefore, they had a great potential to be used as biocontrol agents to effectively manage septoria tritici blotch of wheat (STB).

Elsinochrome phytotoxin production and pathogenicity of Elsinoë arachidis isolates in China.

Peanut scab caused by Elsinoë arachidis is found throughout China’s peanut-growing areas. Elsinochrome produced by E. arachidis is a perylenequinone photosensitive mycotoxin vital to the pathogenic process of the pathogen. In this study, the complex mechanism underlying the regulation of elsinochrome biosynthesis by E. arachidis was investigated based on various nutritional and environmental factors. The initiation of elsinochrome biosynthesis depends on light. E. arachidis produced substantially more quantities of elsinochrome when grown on a semi-synthetic medium (PDA) than when grown on synthetic media with defined ingredients in the presence of light. Elsinochrome accumulation decreased when adjusted with either citrate or phosphate buffers and changing pH suppressed the radical growth. At temperatures ranging from 10°C to 25°C, the production of elsinochrome increased, peaking at 28°C, and it decreased slightly at 30°C. 63 field-collected isolates from China were assessed for the level of elsinochrome production, and pathogenicity analysis was conducted by selecting 12 strains from each 3 of the 4 groups with different levels of elsinochrome production. A direct correlation was observed between elsinochrome production and pathogenicity among the isolates. The results showed elsinochrome biosynthesis to be controlled by E. arachidis and showed elsinochrome to be a vital virulence factor of E. arachidis, required for disease severity.

Genotype‐by‐genotype interactions between an insect and its pathogen

Genotype-by-genotype (G×G) interactions are an essential requirement for the coevolution of hosts and parasites, but have only been documented in a small number of animal model systems. G×G effects arise from interactions between host and pathogen genotypes, such that some pathogen strains are more infectious in certain hosts and some hosts are more susceptible to certain pathogen strains. We tested for G×G interactions in the gypsy moth (Lymantria dispar) and its baculovirus. We infected 21 full-sib families of gypsy moths with each of 16 isolates of baculovirus and measured the between-isolate correlations of infection rate across host families for all pairwise combinations of isolates. Mean infectiousness varied among isolates and disease susceptibility varied among host families. Between-isolate correlations of infection rate were generally less than one, indicating nonadditive effects of host and pathogen type consistent with G×G interactions. Our results support the presence of G×G effects in the gypsy moth-baculovirus interaction and provide empirical evidence that correlations in infection rates between field-collected isolates are consistent with values that mathematical models have previously shown to increase the likelihood of pathogen polymorphism.

Phenotypic Variation in Overwinter Environmental Transmission of a Baculovirus and the Cost of Virulence.

A pathogen's ability to persist in the environment is an ecologically important trait, and variation in this trait may promote coexistence of different pathogen strains. We asked whether naturally occurring isolates of the baculovirus that infects gypsy moth larvae varied in their overwinter environmental transmission and whether this variation was consistent with a trade-off or an upper limit to virulence that might promote pathogen diversity. We used experimental manipulations to replicate the natural overwinter infection process, using 16 field-collected isolates. Virus isolates varied substantially in the fraction of larvae infected, leading to differences in overwinter transmission rates. Furthermore, isolates that killed more larvae also had higher rates of early larval death in which no infectious particles were produced, consistent with a cost of high virulence. Our results thus support the existence of a cost that could impose an upper limit to virulence even in a highly virulent pathogen.

Susceptibility of Adult Cat Fleas (Siphonaptera: Pulicidae) to Insecticides and Status of Insecticide Resistance Mutations at the Rdl and Knockdown Resistance Loci

The susceptibility of 12 field-collected isolates and 4 laboratory strains of cat fleas, Ctenocephalides felis was determined by topical application of some of the insecticides used as on-animal therapies to control them. In the tested field-collected flea isolates the LD50 values for fipronil and imidacloprid ranged from 0.09 to 0.35 ng/flea and 0.02 to 0.19 ng/flea, respectively, and were consistent with baseline figures published previously. The extent of variation in response to four pyrethroid insecticides differed between compounds with the LD50 values for deltamethrin ranging from 2.3 to 28.2 ng/flea, etofenprox ranging from 26.7 to 86.7 ng/flea, permethrin ranging from 17.5 to 85.6 ng/flea, and d-phenothrin ranging from 14.5 to 130 ng/flea. A comparison with earlier data for permethrin and deltamethrin implied a level of pyrethroid resistance in all isolates and strains. LD50 values for tetrachlorvinphos ranged from 20.0 to 420.0 ng/flea. The rdl mutation (conferring target-site resistance to cyclodiene insecticides) was present in most field-collected and laboratory strains, but had no discernible effect on responses to fipronil, which acts on the same receptor protein as cyclodienes. The kdr and skdr mutations conferring target-site resistance to pyrethroids but segregated in opposition to one another, precluding the formation of genotypes homozygous for both mutations.

A method for eliminating bacterial contamination from in vitro moss cultures

• Premise of the study: Bacterial contamination is a major problem in plant tissue culture, resulting in loss of experimental strains or preventing use of field-collected isolates. Here we evaluated an agar embedding method for eliminating bacteria from experimental cultures of the mosses Ceratodon purpureus and Physcomitrella patens.• Methods and Results: We blended moss protonema that had been inoculated with bacteria and embedded the cell fragments in antibiotic-containing, low-concentration agar. The plants were placed in a growth chamber and allowed to grow until the moss grew out of the media. The plants were then transferred to new plates and observed for contamination. The embedding method consistently outperformed standard procedures.• Conclusions: The embedding method places moss in direct contact with antibiotics, arresting bacterial replication and allowing moss to outgrow contamination. We anticipate this method will prove valuable for other plants capable of clonal propagation by blending.

Susceptibility of Cat Fleas (Siphonaptera: Pulicidae) to Fipronil and Imidacloprid Using Adult and Larval Bioassays

The monitoring of the susceptibility offleas to insecticides has typically been conducted by exposing adults on treated surfaces. Other methods such as topical applications of insecticides to adults and larval bioassays on treated rearing media have been developed. Unfortunately, baseline responses of susceptible strains of cat flea, Ctenocephalides felis (Bouchè), except for imidacloprid, have not been determined for all on-animal therapies and new classes of chemistry now being used. However, the relationship between adult and larval bioassays of fleas has not been previously investigated. The adult and larval bioassays of fipronil and imidacloprid were compared for both field-collected isolates and laboratory strains. Adult topical bioassays of fipronil and imidacloprid to laboratory strains and field-collected isolates demonstrated that LD50s of fipronil and imidacloprid ranged from 0.11 to 0.40 nanograms per flea and 0.02 to 0.18 nanograms per flea, respectively. Resistance ratios for fipronil and imidacloprid ranged from 0.11 to 2.21. Based on the larval bioassay published for imidacloprid, a larval bioassay was established for fipronil and reported in this article. The ranges of the LC50s of fipronil and imidacloprid in the larval rearing media were 0.07-0.16 and 0.11-0.21 ppm, respectively. Resistance ratios for adult and larval bioassays ranged from 0.11 to 2.2 and 0.58 to 1.75, respectively. Both adult and larval bioassays provided similar patterns for fipronil and imidacloprid. Although the adult bioassays permitted a more precise dosage applied, the larval bioassays allowed for testing isolates without the need to maintain on synthetic or natural hosts.

Application of in vitro anthelmintic sensitivity assays to canine parasitology: Detecting resistance to pyrantel in Ancylostoma caninum

Resistance of the canine hookworm Ancylostoma caninum to anthelmintic therapy with pyrantel is an emerging problem in canine veterinary practice. Detecting anthelmintic resistance in parasites of pets is problematic because traditional resistance-monitoring techniques used with livestock parasites, such as the faecal egg count reduction test, are often impractical for use in small animals. We used two field-collected isolates of A. caninum in an abbreviated critical trial to test their pyrantel resistance status. The strains showed high-level and low-level resistance, with in vivo pyrantel efficacies of 28% and 71%, respectively. We noted a distinct worm density dependence effect on faecal egg count during the critical trial; egg counts in the dogs containing the low-level resistant isolate were 41% higher 6 days after drug treatment, despite the removal of 71% of the adult worms by the drug treatment. We then assessed four candidate in vitro assays for their ability to detect pyrantel resistance in A. caninum larvae, using these two isolates. The assays included a new format termed the larval arrested morphology assay (LAMA), based on observation of the effects of pyrantel on the body shape adopted by infective stage A. caninum larvae in vitro. Our data suggests that three of these assays, the LAMA, the larval motility assay (LMA), and larval feeding inhibition assay (LFIA) show promise with regards to detection of pyrantel resistance in A. caninum, but the complexity of the LFIA would likely limit its suitability for field studies. In vivo pyrantel efficacies of 28% and 71% in the two A. caninum isolates were associated with a 17-fold shift in the in vitro IC 50 values measured using the LAMA. Further testing with isolates of varying degrees of resistance is required to determine which of these assays is suitable as a rapid in vitro laboratory test for pyrantel resistance in A. caninum. The present study also indicates that potential exists for the novel LAMA or the LMA to be of use in detecting pyrantel resistance in the human hookworms, Necator americanus and Ancylostoma duodenale.

Comparing Methods for Identifying Bacillus Strains Capable of Producing the Antifungal Lipopeptide Iturin A

Lipopeptides represent a unique class of bioactive microbial secondary metabolites, and iturin A shows attractive antibiotic properties among them. This study compares three methods, such as yeast/fungal growth inhibition assay, quantitative high-performance liquid chromatography (HPLC) and polymerase chain reaction (PCR) for identifying a number of Bacillus species that produce iturin A. We examined the feasibility of screening iturin A-producing Bacillus strains by PCR using specific primers for ituD and lpa-14 amplification. Twenty standard strains and 120 field-collected Bacillus spp. isolates were tested in this study. Four B. subtilis and one B. circulan strains from ATCC, and B. amyloliquefaciens B128, a known iturin A producer, exhibited positive results. Of the 120 field-collected isolates, 42 strains were positive. The potential of producing iturin A by these PCR-positive strains were then confirmed by conventional methods such as fungal growth inhibition assay and HPLC analysis. The consistency between results of PCR, HPLC, and fungal growth inhibition assay suggests that the PCR method could be used as an alternative tool for fast screening of iturin A-producing Bacillus strains from the environment. This is the first report of detecting iturin A production from B. circulans.

Determining a Diagnostic Dose for Imidacloprid Susceptibility Testing of Field-Collected Isolates of Cat Fleas (Siphonaptera: Pulicidae)

The susceptibility of four laboratory strains of cat fleas, Ctenocephalides felis (Bouche), to imidacloprid was determined by three different laboratories, by using a standardized bioassay protocol. The probit lines generated by the different laboratories were very similar, with LC50 values ranging from 0.32 to 0.81 ppm. Based on these data, a diagnostic dose (DD) of 3 ppm imidacloprid in larval rearing media was provisionally identified for detecting shifts in tolerance, possibly as a consequence of incipient imidacloprid resistance. None of the larvae from the susceptible laboratory strains survived the DD. Eighteen field-collected isolates were evaluated for their susceptibility to imidacloprid and to validate a DD of 3 ppm. Probit lines from 18 field-collected isolates were very similar, with LC50 values ranging from 0.14 to 1.52 ppm. When exposed to the DD, between 3 and 10% of the exposed larvae emerged as adults from only three of the 18 isolates. All other field isolates gave 100% mortality at the DD. Under the criteria established (>5% survivorship at 3 ppm), two isolates would be established on mammalian hosts and more extensive tests conducted to exclude or confirm the presence of resistance. The DD of 3 ppm is robust enough to eliminate most of the susceptible isolates collected until today, yet low enough to identify possible isolates for further testing.

Determining a Diagnostic Dose for Imidacloprid Susceptibility Testing of Field-Collected Isolates of Cat Fleas (Siphonaptera: Pulicidae)

The susceptibility of four laboratory strains of cat fleas, Ctenocephalides felis (Bouché), to imidacloprid was determined by three different laboratories, by using a standardized bioassay protocol. The probit lines generated by the different laboratories were very similar, with LC50 values ranging from 0.32 to 0.81 ppm. Based on these data, a diagnostic dose (DD) of 3 ppm imidacloprid in larval rearing media was provisionally identified for detecting shifts in tolerance, possibly as a consequence of incipient imidacloprid resistance. None of the larvae from the susceptible laboratory strains survived the DD. Eighteen field-collected isolates were evaluated for their susceptibility to imidacloprid and to validate a DD of 3 ppm. Probit lines from 18 field-collected isolates were very similar, with LC50 values ranging from 0.14 to 1.52 ppm. When exposed to the DD, between 3 and 10% of the exposed larvae emerged as adults from only three of the 18 isolates. All other field isolates gave 100% mortality at the DD. Under the criteria established (>5% survivorship at 3 ppm), two isolates would be established on mammalian hosts and more extensive tests conducted to exclude or confirm the presence of resistance. The DD of 3 ppm is robust enough to eliminate most of the susceptible isolates collected until today, yet low enough to identify possible isolates for further testing.

Cloning and characterization of the mating type ( MAT) locus from Ascochyta rabiei (teleomorph: Didymella rabiei) and a MAT phylogeny of legume-associated Ascochyta spp.

Degenerate primers designed to correspond to conserved regions of the high mobility group (HMG) protein encoded by the MAT1-2 gene of Cochliobolus heterostrophus, Cochliobolus sativus, and Alternaria alternata were used to amplify the portion of the sequence corresponding to the HMG box motif from Ascochyta rabiei (teleomorph: Didymella rabiei). A combination of TAIL and inverse PCR extended the MAT1-2 sequence in both directions, then primers designed to MAT1-2 flanking DNA were used to amplify the entire MAT1-1 idiomorph. MAT1-1 and MAT1-2 idiomorphs were 2294 and 2693 bp in length, respectively, and each contained a single putative open reading frame (ORF) and intron similar to MAT loci of other loculoascomycete fungi. MAT genes were expressed at high levels in rich medium. MAT-specific PCR primers were designed for use in a multiplex PCR assay and MAT-specific PCR amplicons correlated perfectly to mating phenotype of 35 ascospore progeny from a cross of MAT1-1 by MAT1-2 isolates and to the mating phenotype of field-collected isolates from diverse geographic locations. MAT-specific PCR was used to rapidly determine the mating type of isolates of A. rabiei sampled from chickpea fields in the US Pacific Northwest. Mating type ratios were not significantly different from 1:1 among isolates sampled from two commercial chickpea fields consistent with the hypothesis that these A. rabiei populations were randomly mating. The mating type ratio among isolates sampled from an experimental chickpea field where asexual reproduction was enforced differed significantly from 1:1. A phylogeny estimated among legume-associated Ascochyta spp. and related loculoascocmycete fungi using sequence data from the nuclear ribosomal internal transcribed spacer (ITS) demonstrated the monophyly of Ascochyta/Didymella spp. associated with legumes but was insufficiently variable to differentiate isolates associated with different legume hosts. In contrast, sequences of the HMG region of MAT1-2 were substantially more variable, revealing seven well-supported clades that correlated to host of isolation. A. rabiei on chickpea is phylogenetically distant from other legume-associated Ascochyta spp. and the specific status of A. rabiei, A. lentis, A. pisi, and A. fabae was confirmed by the HMG phylogeny

Biological characteristics of praziquantel-resistant and -susceptible isolates of Schistosoma mansoni.

If there is a change in the biological characteristics of schistosomes associated with the development of resistance to praziquantel, this could affect the transmission and pathology of the diseases they cause. To investigate this possibility, the host-parasite relationships of five praziquantel-resistant and five praziquantel-susceptible isolates of Schistosoma mansoni were compared in Biomphalaria glabrata snails and outbred CD(1) albino mice. Whether praziquantel-resistant or -susceptible, all the laboratory-selected isolates gave similar results in B. glabrata. However, the snails infected with any of three field-collected isolates from Senegal, each of which has been shown to be praziquantel-resistant, shed fewer cercariae and survived longer than the snails infected with the drug-susceptible or laboratory-selected, drug-resistant isolates. There were no differences between isolates in terms of their infectivity to mice. However, mice infected with any of four of the five drug-resistant isolates shed more eggs in their faeces than mice carrying the drug-susceptible parasites, and mice infected with any of the resistant isolates also had larger numbers of eggs in their tissues. Although granuloma sizes were generally similar for all isolates, the praziquantel-resistant isolates may be more pathogenic in mice than the susceptible ones because of their relatively high egg productions.

Resistance of Botryosphaeria dothidea from Pistachio to Iprodione.

Out of 79 field-collected isolates of Botryosphaeria dothidea, causal agent of panicle and shoot blight of California pistachio, 1 had low resistance to the dicarboximide fungicide iprodione (effective fungicide concentration to inhibit 50% of mycelial growth [EC50] = 2.726 μg/ml), whereas all the other isolates were sensitive (EC50 < 1 μg/ml). B. dothidea isolates readily developed resistance to iprodione in vitro. Furthermore, these in vitro-derived iprodione-resistant (IR) isolates retained high virulence on pistachio. The EC50 values for these IR isolates were greater than 25 μg/ml, whereas the EC50 values for the original wild-type counterparts were less than 1 μg/ml. Iprodione resistance significantly declined when these IR isolates were propagated on pistachio leaves in the absence of the fungicide. IR isolates also were resistant to vinclozolin, another dicarboximide fungicide, but sensitive to tebuconazole and benomyl. IR isolates showed an increase in osmotic sensitivity on media amended with NaCl. Applications of iprodione at 500 μg/ml, which is effective against naturally sensitive isolates, failed to control disease caused by IR isolates in both the laboratory and greenhouse. The results indicate that, although naturally occurring IR isolates of B. dothidea may be rare in California pistachio orchards, the fungus readily develops resistance to iprodione in vitro and, more importantly, retains high levels of virulence on pistachio.

Multiplex PCR screening to detect cry9 genes in Bacillus thuringiensis strains.

An extended PCR method was established to rapidly identify and classify Bacillus thuringiensis strains containing cry (crystal protein) genes toxic to lepidopteran, coleopteran, and dipteran pests (Ben-Dov et al., Appl. Environ. Microbiol. 63:4883-4890, 1997). To optimize identification of all reported cry genes, this methodology needs a complete PCR set of primers. In the study reported here, a set of universal (Un9) and specific primers for multiplex rapid screening for all four known genes from the cry9 group was designed. PCR analyses were performed for cry9 genes on 16 standard strains and 215 field isolates of B. thuringiensis. Among the standard strains, only B. thuringiensis subsp. aizawai HD-133, which harbors cry1 and cry2 genes, was positive with Un9 but negative to all four specific primers for cry9 genes. DNA of 22 field-collected isolates was also found to be positive with Un9. These isolates were classified into three cry9 profiles using specific primers; all of them harbor cry1 and cry2. This newly designed set of primers complements the existing PCR methodology for most currently known cry genes.

Diversity ofPseudomonas fuscovaginaeand Other Fluorescent Pseudomonads Isolated from Diseased Rice

Fluorescent pseudomonads associated with sheath brown rot of rice were isolated from rice at low and high elevations in various geographic areas and analyzed for pathogenicity and phenotypic and genetic diversity by biochemical tests and restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified 16S-rDNA genes. Standard strains of various Pseudomonas species of rRNA homology group I were analyzed along with field-collected isolates. Biochemical characterization revealed 23 groups characterized by different nutritional features. Nineteen of thirty-five rice isolates were identified as P. fuscovaginae (biochemical group G1). Using PCR-RFLP, 25 composite 16S-rDNA haplotypes were found. Parsimony analysis revealed that the P. fuscovaginae isolates were distributed in four 16S-rDNA haplotypes, but they formed a cluster that differentiated them from other rRNA group I Pseudomonas species. All field isolates were pathogenic on rice when introduced by injection. Inoculation of rice without wounding at the booting stage showed that only the four haplotypes of P. fuscovaginae and isolates with haplotypes 7, 9, and 17 were pathogenic. Thus, populations of rice-associated fluorescent pseudomonads comprised both phylogenetically different pathogenic isolates and opportunistic isolates pathogenic on rice only after wounding.

The California Population of Agaricus bisporus Comprises at Least Two Ancestral Elements

Genotypes of a sample of field-collected heterokaryon isolates of Agaricus bisporus from California were compared with those of a comprehensive sample of cultivar isolates that are likely to be of European ancestry. Isolate genotypes were determined for 14 nuclear DNA restriction fragment length polymorphism markers and seven allozyme markers, and were compared in a pairwise fashion to produce a matrix of genotypic dissimilarities between all isolates. This matrix was evaluated with regard to the affinities of individual California isolates, both among themselves and with a reconstructed population ancestral to the cultivars. Most isolates from California fell into one of two apparent groups; these either: 1) had high affinity with the cultivars or 2) differed substantially from the cultivars and aggregated in a distinct, cypress-associated Californian group. A few isolates had an intermediate character. This result, supported by habitat correlations and the distribution of characteristic mitochondrial DNA types, indicates that the California population presently comprises introduced cultivars, indigenous cypress-associated strains, and a less distinct group that could include spontaneous hybrids between these two ancestral elements, or strains with other affinities. If hybridization is indeed occurring, the two major ancestral elements have introgressed only slightly. Agaricus bisporus (Lange) Imbach, the familiar Button Mushroom of commerce (for nomenclatural debate see Kerrigan 1987; Malloch et al. 1987), is now cultivated on six continents. Little is known, however, about populations of this species. Historical evidence strongly indicates that most or all cultivar lines were selected, over the last three centuries, from wild mushrooms collected in Europe, often found in association with accumulations of horse manure or other agricultural wastes (Falconer 1891; Robinson 1870). Genetically, cultivar isolates form a relatively homogeneous group, which also suggests that they were originally drawn from a single population or gene pool (Kerrigan 1990; May and Royse 1982; Royse and May 1982a). Cultivars are known to have become naturalized in North America, based on analysis of genetic markers in field-collected isolates (Kerrigan and Ross 1989, and below). Dispersal of germ plasm has apparently been exclusively unidirectional, from Europe; to our knowledge no foreign germ plasm had been sent to Europe prior to our distribution of research cultures beginning in 1989. As a result of this asymmetry, a sample of the (European) A. bisporus population from which the cultivar stocks were drawn can plausibly be reconstructed from extant cultivars. In contrast, field samples from almost any other region could include elements having either cultivar, indigenous, or hybrid ancestry. The possible existence of indigenous, non-European populations of A. bisporus, and even the status of the organism as a natural have been called into question (May and Royse, 1982; Raper et al. 1972). Morphologically, the species concept is well delimited, as this is the only known species of Agaricus with predominately bisporic basidia. The possibilities of multiple independent origins of this trait, or of divergence into reproductively isolated biological species, have not previously been addressed. Evidence from six allozyme loci for the existence in California of an indigenous, non-cultivar population of A. bisporus was presented by Kerrigan and Ross (1989). Studies of A. bisporus populations are complicated by the unpredictability of the occurrence of the ephemeral sporocarps, by which individual mycelia are located, and by the fact that over the great majority of its range, the species is only very occasionally encountered. This suggests that population densities may typically be low, relative to many