Geographical distribution of milky disease bacteria in the eastern United States based on phylogeny

A phylogenetic grouping of 48 different isolates of milky disease bacteria isolated in the United States was determined using genomic RFLP analysis and 16S rDNA sequence comparison. A clear distinction between Paenibacillus popilliae isolates and Paenibacillus lentimorbus isolates was evident from the results of each procedure. The P. popilliae isolates segregated into two phylogenetic groups and the P. lentimorbus isolates segregated into three phylogenetic groups. In the United States, P. popilliae group 1 was generally isolated from insects collected west of the Appalachian Mountains. P. popilliae group 2 was only isolated from insects collected east of the Appalachian Mountains. P. lentimorbus groups 1 and 2 were obtained from insects collected west and south of the Appalachians. P. lentimorbus group 3 was identified in insects collected east of the mountains. From five different locations in Connecticut, 12 milky disease bacterial isolates were classified as P. popilliae and three were classified as P. lentimorbus. Except for one isolate, all P. popilliae isolates were of phylogenetic group 2. The three P. lentimorbus strains were isolated from diseased insects that had been collected from a localized area in the state. These three strains formed a separate phylogenetic grouping (i.e., group 3) of P. lentimorbus and, based on 16S rDNA sequence comparisons, were most similar to the newly identified P. lentimorbus Semadara strain recently isolated in Japan. All milky disease bacteria that had been isolated from commercially available insecticide preparations were identified as P. popilliae group 1.     

Milky disease bacteria

A comparative study was made of all available milky-disease species and strains that have been isolated around the world from beetle larvae (family Scarabaeidae). Included in the study were Bacillus popilliae Dutky, B. lentimorbus Dutky, and B. lentimorbus var. maryland from the United States; B. euloomarahae Beard and B. lentimorbus var. australis Beard from Australia; B. fribourgensis Wille from Switzerland; and New Zealand milky disease (Dumbleton). The organisms were classified into three groups: (i) those containing parasporal bodies, including B. popilliae Dutky, B. fribourgensis Wille, and New Zealand milky disease (Dumbleton); (ii) those without a visible parasporal body and with spore morphology similar to B. lentimorbus Dutky, including B. lentimorbus var. australis Beard; and (iii) those with very tiny spores and no parasporal body, including B. euloomarahae Beard and B. lentimorbus var. maryland. All available milky-disease species and strains were cultivated in vitro on Brain Heart Infusion Agar plates. However, the most fastidious organisms-B. euloomarahae and B. lentimorbus var. maryland-could not be grown until they were passed through a life cycle in larvae of a large scarabaeid beetle infesting rotting wood. Then they remained stable for only one or two subcultures. All the milky-disease organisms produced larger cells in vitro than they did in vivo. The pattern of sugar fermentations was similar for all milky-disease species. It appears that there is a very low percentage of strains of B. popilliae, B. lentimorbus, and the other milky-disease organisms that have the inherent genetic makeup to permit them to sporulate on artificial media, if conditions are favorable. Among these conditions are a sufficiently high cell population and a reduced oxygen tension. Spores produced in vitro may have a low virulence via the normal ingestion pathway, even though they show apparent virulence when injected directly into the hemocoel.     

Susceptibility of the taro beetle, Papuana uninodis (Coleoptera, Scarabaeidae) to two new Bacillus popilliae isolates from Papuana spp

Two morphological types of Bacillus popilliae, causal agent of the milky disease, have been isolated from taro beetles (Papuana spp, Coleoptera: Scarabaeidae). B. popilliae from P. woodlarkiana woodlarkiana (Papua New Guinea) was a type A1 with a small sporangium (4.1 x 1.6 microm) and a large spore (2.1 x 1.4 microm) and parasporal body (1.8 x 1.2 microm) that sometimes overlap. B. popilliae from P. uninodis and P. woodlarkiana laevipennis (Solomon Islands) was a type B2 with a small sporangium (2.8 x 1.3 microm), a small eccentric spore (1.1 x 0.7 microm), and no parasporal body. The infectivity of these B. popilliae to Papuana uninodis larvae was compared with two B. popilliae samples from Popillia japonica in injection tests. The hemolymph of P. uninodis supported the germination and growth of isolates from Papuana and P. japonica. Results were similar in third instars and adults. Highest infection (spores present) and mortality was caused by the isolates from Papuana: mortality reached almost 100% 4 weeks after injection of the B2 type B. popilliae with 40% of larvae and 52% of adults infected. Injection of type A1 caused lower mortality but a similar percentage infected. Of two A1 B. popilliae from P. japonica, one caused a mortality comparable to type A1 from Papuana but lower infection; an older isolate resulted in low mortality and only one infected larva. B. popilliae type A1 from P. woodlarkiana was produced in the Solomon Islands by injection of spores in P. uninodis. Thirty four percent of the injected larvae and 31% of the adults produced spores with an average yield of 3.2 and 0.8 x 10(9) spores/insect, respectively. Oral application of a single dose of 10(7) spores of the B. popilliae isolates from P. uninodis or P. japonica did not cause infection and similarly inoculation of the food with spores of B. popilliae type B2 did not result in infections. However, when different rates were applied to the food of second- and third-instar P. uninodis, the B. popilliae type A1 from P. woodlarkiana caused up to 15% infection and concentration-related mortality.     

Novel cry gene from Paenibacillus lentimorbus strain Semadara inhibits ingestion and promotes insecticidal activity in Anomala cuprea larvae

A positive clone was selected from a library of total cell DNA of Paenibacillus lentimorbus strain Semadara that reacted with an antiserum that was raised against parasporal crystal proteins produced by this strain. The positive clone had a DNA insert containing two whole cry genes (cry43Aa1, cry43Ba1), one partial cry gene (cry43-like), and three smaller genes located upstream. Eight blocks that are conserved in the Cry proteins of Bacillus thuringiensis [Microbiol. Mol. Biol. Rev. 62 (1998) 775] were detected in their deduced amino acid sequences. The Escherichia coli transformant expressing cry43Aa1 caused inhibition of ingestion and 90% mortality in the first stadium larvae of Anomala cuprea. A low concentration of sporangia mixed with the transformant expressing cry43Aa1 easily infected the larvae of A. cuprea. The protein of approximately 150 kDa produced by the transformants expressing the cry genes reacted with antiserum specific for the parasporal crystal proteins. Southern hybridization analysis demonstrated that the cry genes were located on the chromosomal DNA of this strain, which possessed at least four cry genes.     

Cloning and analysis of the first cry gene from Bacillus popilliae.

An 80-kDa parasporal crystal protein was detected in protein extracts of sporangia of Bacillus popilliae isolated from a diseased larva of the common cockchafer (Melolontha melolontha L.). Amino acid analysis of tryptic peptides revealed significant homology to the Cry2Aa endotoxins of Bacillus thuringiensis. The gene cryBP1 (cry18Aa1), which codes for the parasporal crystal protein, was found in a putative cry operon on the bacterial chromosome, which contains at least one further (smaller) open reading frame, orf1. The 706-amino-acid-long CryBP1 (Cry18Aa1) protein has a predicted molecular mass of 79 kDa and shows about 40% sequence identity to the Cry2 polypeptides of B. thuringiensis. In the light of published observations which suggest that the parasporal crystal proteins of B. popilliae are slightly toxic to their grub hosts, we propose the following survival strategy of B. popilliae. As an obligate pathogen of grubs, B. popilliae germinates in the gut of a grub and the parasporal crystal proteins are released and activated. The activated protein does not cause colloid osmotic lysis but instead damages the gut wall somehow to allow the vegetative cells to enter the hemolymph more easily. By becoming a parasite, B. popilliae can continue to proliferate efficiently while the living grub provides a food supply. This process is in contrast to that of B. thuringiensis, which rapidly kills the insect and is then limited to growth on the larval carcass.     

Molecular Characterization of Subtype H11N9 Avian Influenza Virus Isolated from Shorebirds in Brazil

Migratory aquatic birds play an important role in the maintenance and spread of avian influenza viruses (AIV). Many species of aquatic migratory birds tend to use similar migration routes, also known as flyways, which serve as important circuits for the dissemination of AIV. In recent years there has been extensive surveillance of the virus in aquatic birds in the Northern Hemisphere; however in contrast only a few studies have been attempted to detect AIV in wild birds in South America. There are major flyways connecting South America to Central and North America, whereas avian migration routes between South America and the remaining continents are uncommon. As a result, it has been hypothesized that South American AIV strains would be most closely related to the strains from North America than to those from other regions in the world. We characterized the full genome of three AIV subtype H11N9 isolates obtained from ruddy turnstones (Arenaria interpres) on the Amazon coast of Brazil. For all gene segments, all three strains consistently clustered together within evolutionary lineages of AIV that had been previously described from aquatic birds in North America. In particular, the H11N9 isolates were remarkably closely related to AIV strains from shorebirds sampled at the Delaware Bay region, on the Northeastern coast of the USA, more than 5000 km away from where the isolates were retrieved. Additionally, there was also evidence of genetic similarity to AIV strains from ducks and teals from interior USA and Canada. These findings corroborate that migratory flyways of aquatic birds play an important role in determining the genetic structure of AIV in the Western hemisphere, with a strong epidemiological connectivity between North and South America.     

Multiple cryptic species with divergent substrate affinities in the Serpula himantioides species complex

Serpula himantioides is a widespread saprotrophic morphospecies mainly colonising coniferous wood in nature, but it appears frequently in buildings as well. From an earlier study, it is known that at least three divergent lineages occur within the S. himantioides species complex. In this study, a broader sample of S. himantioides isolates has been analysed by multi-locus sequencing, including new isolates from Asia, North and South America. Altogether five phylogenetical species (PS1-5) were detected, all recognised across independent gene phylogenies. A new southern South American phylogenetic species (PS1) was found, representing an early diverging lineage within the S. himantioides species complex. The two closely related PS2 and PS3 lineages included isolates from North America only, and PS4 was also dominated by North American isolates. Most of the investigated isolates (76%) clustered into PS5, a lineage that has been found on most continents, including North America. Overall, little phylogeographical structure was found in PS5, indicating frequent and recent long-distance dispersal events within this widespread lineage. Our analyses indicate that South and North America are the centres of divergence for the S.?himantioides species complex. Some of the lineages seem adapted to various substrates, but PS5 is able to decay a wide array of angiosperms and gymnosperms, which may have facilitated the spread of this lineage throughout the world.     

Mitochondrial DNA phylogeny of the woodpecker genus Veniliornis (Picidae, Picinae) and related genera implies convergent evolution of plumage patterns

The woodpecker genus Veniliornis comprises 12 species, all restricted to the New World tropics. The seemingly distantly related genus Picoides is broadly distributed in Eurasia and North America with two putative species, P. lignarius and P. mixtus , occurring in South America. The two genera are clearly distinct with respect to general plumage colouration and patterning as well as habitat utilization and thus traditionally have been placed in different tribes. Phylogenetic analyses of mtDNA sequences from the COI and cyt b genes indicated that both genera are reciprocally paraphyletic. The two South American species of Picoides belong to a clade comprising most species of Veniliornis , but V. fumigatus of Central and north-western South America belongs to a clade comprising species of Picoides . The mtDNA tree also indicated that Veniliornis is not closely related to the genus Piculus, as is implicit in current classifications. Misclassifications involving Veniliornis at both the generic and tribal levels appear to result from convergent evolution of plumage traits in specific forest types. We infer that the common ancestor of Veniliornis entered South America approximately at the time the Isthmus of Panama was formed, and diversification within South America was rapid.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 87 , 611–624.     

Phylogeny of South American Bufo (Anura: Bufonidae) inferred from combined evidence

Despite the considerable research that has focused on the evolutionary relationships and biogeography of the genus Bufo, an evolutionary synthesis of the entire group has not yet emerged. In the present study, almost 4 kb of DNA sequence data from mitochondrial (12S, tRNA^Val, and 16S) and nuclear (POMC; Rag-1) genes, and 83 characters from morphology were analysed to infer a phylogeny of South American toads. Phylogenies were reconstructed with parsimony and maximum likelihood and Bayesian model-based methods. The results of the analysis of morphological data support the hypothesis that within Bufo , some skull characters (e.g. frontoparietal width), correlated with the amount of cranial ossification, are prone to homoplasy. Unique and unreversed morphological synapomorphies are presented that can be used to diagnose recognized species groups of South American toads. The results of all phylogenetic analyses support the monophyly of most species groups of South American Bufo . In most DNA-only and combined analyses, the South American (minus the B. guttatus and part of the ' B. spinulosus ' groups), North American, Central American, and African lineages form generally well-supported clades: ((((((((South America) (North America + Central America)) Eurasia) Africa) Eurasia) South America) West Indies) South America). This result confirms and extends prior studies recovering South American Bufo as polyphyletic. The biogeographical results indicate that: (1) The origin of Bufo predates the fragmentation of Gondwana; (2) Central and North American species compose the sister group to a large, 'derived' clade of South American Bufo ; and (3) Eurasian species form the sister group to the New World clade.  © 2006 The Linnean Society of London, Zoological Journal of the Linnean Society , 2006, 146 , 407–452.     

Characterization of Paenibacillus popilliae rRNA operons

The terminal 39 nucleotides on the 3' end of the 16S rRNA gene, along with the complete DNA sequences of the 5S rRNA, 23S rRNA, tRNA(Ile), and tRNA(Ala) genes were determined for Paenibacillus popilliae using strains NRRL B-2309 and Dutky 1. Southern hybridization analysis with a 16S rDNA hybridization probe and restriction-digested genomic DNA demonstrated 8 copies of the 16S rRNA gene in P. popilliae strains KLN 3 and Dutky 1. Additionally, the 23S rRNA gene in P. popilliae strains NRRL B-2309, KLN 3, and Dutky 1 was shown by I-CeuI digestion and pulsed-field gel electrophoresis of genomic DNA to occur as 8 copies. It was concluded that these 3 P. popilliae strains contained 8 rrn operons. The 8 operon copies were preferentially located on approximately one-half of the chromosome and were organized into 3 different patterns of genes, as follows: 16S-23S-5S, 16S-ala-23S-5S, and 16S-5S-ile-ala-23S-5S. This is the first report to identify a 5S rRNA gene between the 16S and 23S rRNA genes of a bacterial rrn operon. Comparative analysis of the nucleotides on the 3' end of the 16S rRNA gene suggests that translation of P. popilliae mRNA may occur in Bacillus subtilis and Escherichia coli.     

Rapid global spread of two aggressive strains of a wheat rust fungus

Rust fungi can overcome the effect of host resistance genes rapidly, and spores can disperse long distance by wind. Here we demonstrate a foreign incursion of similar strains of the wheat yellow rust fungus, Puccinia striiformis f. sp. tritici , in North America, Australia and Europe in less than 3 years. One strain defined by identity at 15 virulence loci and 130 amplified fragment length polymorphism (AFLP) fragments was exclusive to North America (present since 2000) and Australia (since 2002). Another strain of the same virulence phenotype, but differing in two AFLP fragments, was exclusive to Europe (present since 2000–2001) as well as Western and Central Asia and the Red Sea Area (first appearance unknown). This may be the most rapid spread of an important crop pathogen on the global scale. The limited divergence between the two strains and their derivatives, and the temporal–spatial occurrence pattern confirmed a recent spread. The data gave evidence for additional intercontinental dispersal events in the past, that is, many isolates sampled before 2000 in Europe, North America and Australia had similar AFLP fingerprints, and isolates from South Africa, which showed no divergence in AFLP, differed by only two fragments from particular isolates from Central Asia, West Asia and South Europe, respectively. Previous research has demonstrated that isolates of the two new strains produced up to two to three times more spores per day than strains found in USA and Europe before 2000, suggesting that increased aggressiveness at this level may accelerate global spread of crop pathogens.     

Phytogeography of the trees of the Osa Peninsula,Costa Rica

A phytogeographic analysis of the distributions of 454 species of trees native to the Osa Peninsula in 22 families revealed that 4.8% of the species are endemic to the Osa Peninsula and the adjacent mainland of Costa Rica. However, nearly one-fourth of the species might be regionally endemic to Central-South Mesoamerica (Costa Rica, Nicaragua, and Panama). Our sample suggests that 53.6% of the species occur in some part of Mesoamerica and sometimes range into northwestern South America, and that 44.5% of the species have wide distributions throughout tropical America. There is a strong affinity with the flora of northwestern South America, with 46.2% of the species on the Osa also found there. In addition, 50.6% of the tree species on the Osa occur on both the Atlantic and Pacific slopes of Central America or, if they reach South America, are sometimes found on both sides of the Andes. Major contributions to the tree flora of the Osa have been made by species arriving in the Osa by 1) dispersal from South and North America to islands in proto Central America before the formation of a dry-land connection between the two continents, and 2) migration from South America and North America after the closure of the Panamanian isthmus was made. This analysis demonstrates the importance of the Osa as a regional refuge for protecting species with distributions limited to the Osa and parts of Panama, Costa Rica, or Nicaragua. The Osa is also important because it harbors the last expanse of tropical wet forest on the Pacific slope of Central America large enough to ensure the survival of the Central American populations of widely distributed plants and animals.     

Classification of Histoplasma capsulatum isolates by restriction fragment polymorphisms

Twenty isolates of the dimorphic, pathogenic fungus Histoplasma capsulatum were divided into three classes based on comparisons of restriction enzyme digests of their mitochondrial DNA and rDNA. The majority of isolates, including most North American strains and the African H. capsulatum var. duboisii variants, belong to class 2. Isolates from Central America and South America make up class 3. The attenuated Downs strain is the only member of class 1.     

Diversity and Distributional Patterns of Neotropical Freshwater Copepods (Calanoida: Diaptomidae)

The distributional patterns and diversity of the diaptomid calanoid copepods were analysed to assess the faunistic affinity of North and South America with respect to Mexico and Central America. In the Neotropical region, the most speciose genera of Diaptomidae are Leptodiaptomus and Mastigodiaptomus. The former genus is a Nearctic form, and Mastigodiaptomus is Neotropical. Based on the current distribution of their diversity, it is probable that these genera radiated into Mexico and Central America from North America and the insular Caribbean, respectively. Arctodiaptomus dorsalis is a primarily Palaearctic taxon, it is widely distributed between North and Central America. This species probably radiated in the Americas as a Tethyan derivate. Prionodiaptomus is the only member of the highly diverse South American diaptomid fauna that has expanded beyond the subcontinent. Despite the high diversity present in South America, its influence in Mexico and Central America appears to be weak; this is probably a consequence of the geologically recent union of the two main subcontinental landmasses. Mexico shares 33% of its species with NA, and no species are shared between NA and SA. For the Diaptomidae, the Nearctic influence is strongest in Mexico. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Sesquiterpene lactones in various populations of Parthenium hysterophorus

Samples of 31 populations of Parthenium hyserophorus from varius areas of its world distribution were examined for their sesquiterpene lactone composition. On the basis of the occurrence of sesquiterpene lactones the samples were divided into seven chemical types. The most common is a type represented by plants containing parthenin as a major sesquiterpene lactone, and coronopilin and tetraneurin-A. All samples from North and Central America, Venezuela, South Africa, India, Australia, and one sample from Jamaica belong to this type. This suggests that P. hysterophorus recently introduced to South Africa, India and Australia originates in North and (or) Central America. On the other hand, there is a great diversity among examined South American populations. Plants from these populations usually contain hymenin which is their major sesquiterpene lactone. Some populations may also contain coronopilin, hysterin, and dihydroisoparthenin. A high diversity in the sesquiterpene lactone chemistry and the morphological differences between individual South American populations of this species indicate the possible existence of several taxa.     

Biogeography of mutualistic fungi cultivated by leafcutter ants

Leafcutter ants propagate co‐evolving fungi for food. The nearly 50 species of leafcutter ants (Atta, Acromyrmex) range from Argentina to the United States, with the greatest species diversity in southern South America. We elucidate the biogeography of fungi cultivated by leafcutter ants using DNA sequence and microsatellite‐marker analyses of 474 cultivars collected across the leafcutter range. Fungal cultivars belong to two clades (Clade‐A and Clade‐B). The dominant and widespread Clade‐A cultivars form three genotype clusters, with their relative prevalence corresponding to southern South America, northern South America, Central and North America. Admixture between Clade‐A populations supports genetic exchange within a single species, Leucocoprinus gongylophorus. Some leafcutter species that cut grass as fungicultural substrate are specialized to cultivate Clade‐B fungi, whereas leafcutters preferring dicot plants appear specialized on Clade‐A fungi. Cultivar sharing between sympatric leafcutter species occurs frequently such that cultivars of Atta are not distinct from those of Acromyrmex. Leafcutters specialized on Clade‐B fungi occur only in South America. Diversity of Clade‐A fungi is greatest in South America, but minimal in Central and North America. Maximum cultivar diversity in South America is predicted by the Kusnezov–Fowler hypothesis that leafcutter ants originated in subtropical South America and only dicot‐specialized leafcutter ants migrated out of South America, but the cultivar diversity becomes also compatible with a recently proposed hypothesis of a Central American origin by postulating that leafcutter ants acquired novel cultivars many times from other nonleafcutter fungus‐growing ants during their migrations from Central America across South America. We evaluate these biogeographic hypotheses in the light of estimated dates for the origins of leafcutter ants and their cultivars.     

Presence of N6-methyladenine in GATC sequences of Bacillus popilliae and Bacillus lentimorbus KLN2.

Nine strains of Bacillus popilliae and Bacillus lentimorbus KLN2 contain N6-methyladenine in GATC sequences, as determined by using the restriction enzymes MboI and DpnI. Among eight other Bacillus species examined, all, except one strain of Bacillus brevis (ATCC 9999), lacked adenine methylation in GATC. A methylase with Escherichia coli dcm site specificity was not present in any of the Bacillus species studied.     

Genetic structure of natural populations of the nitrogen-fixing bacterium Rhizobium meliloti.

The genetic structure of populations of the symbiotic nitrogen-fixing soil bacterium Rhizobium meliloti was examined by analysis of electrophoretically demonstrable allelic variation in 14 metabolic, presumably chromosomal, enzyme genes. A total of 232 strains were examined, most of which were isolated from southwest Asia, where there is an unsurpassed number of indigenous host species for R. meliloti. The collection consisted of 115 isolates recovered from annual species of Medicago in Syria, Turkey, and Jordan; 85 isolates cultured from two perennial species of Medicago (M. sativa [alfalfa] and M. falcata) in northern Pakistan and Nepal; and 32 isolates collected at various localities in North and South America, Europe, South Africa, New Zealand, and Australia, largely from M. sativa. Fifty distinctive multilocus genotypes (electrophoretic types [ETs]) were identified, and cluster analysis revealed two primary phylogenetic divisions separated at a genetic distance of 0.83. By the criterion of genetic differentiation conventionally applied in defining species limits among members of the family Enterobacteriaceae and certain other bacteria, the two primary divisions of R. meliloti represent distinct evolutionary species. Division A included 35 ETs represented by 209 strains from the eastern Mediterranean basin, northern Pakistan, Nepal, and various other localities worldwide. This division contained the nine commercial alfalfa inoculant strains examined. Division B included 15 ETs represented by 23 isolates, 21 of which were isolated from annual medic species growing in previously uninoculated soils in the eastern Mediterranean basin. The two remaining strains in division B, both representing the same ET, were isolated in the United States and Australia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular probes of phylogeny and biogeography in toads of the widespread genus Bufo

Genetic relationships among 25 species of Central and South American Bufo and among representative North, Central, and South American, Asian, and African Bufo were probed, using the quantitative immunological technique of microcomplement fixation (MC'F) which indicated a clear separation of North, Central, and South American lineages of Bufo. The South American lineage likely diverged from the Central and North American lineages in the Eocene; the latter two lineages diverged later, probably in the mid-Oligocene. Some species groups of South American toads, defined on the basis of traditional morphological studies, are genetically quite similar within groups, whereas others are genetically divergent. The amount of albumin evolution does not appear to parallel the amount of karyotypic, morphological, ecological, or behavioral evolution documented. Comparisons suggest that the African lineages separated from the American and Asian lineages in the late Cretaceous, corresponding to the time of the final separation of Gondwanaland, the southern supercontinent including the modern continents of South America, Africa, Australia, Antarctica, and India. The Asian lineages diverged from the lineage giving rise to all of the American species in the early Paleocene.