Corticioid basidiomycetes associated with bark beetles, including seven new Entomocorticium species from North America and Cylindrobasidium ipidophilum, comb. nov.

Seven new Entomocorticium species (Peniophoraceae) are described based on morphology and phylogenetic analyses. Along with the type species (E. dendroctoni), Entomocorticium comprises eight species of nutritional symbionts of pine bark beetles in North America. Entomocorticium cobbii is the mycangial associate of the southern pine beetle, Dendroctonus frontalis, and E. parmeteri is the mycangial associate of the western pine beetle, D. brevicomis. Entomocorticium whitneyi, E. portiae, E. kirisitsii, E. oberwinkleri and the previously described E. dendroctoni have been isolated from galleries of D. ponderosae and D. jeffreyi in western North America. Entomocorticium sullivanii forms an ambrosia-like layer of basidia and basidiospores in the pupal chambers of Ips avulsus in the southeastern USA. Entomocorticium is phylogenetically placed within Peniophora, a corticioid genus of wood decay fungi with wind-dispersed basidiospores. At least four species of Entomocorticium produce basidiospores on basidia with reduced sterigmata that apparently do not forcibly discharge basidiospores. Another basidiomycete, Gloeocystidium ipidophilum, was described from Ips typographus galleries in Europe, but it is phylogenetically and taxonomically placed in another genus of wood decay fungi as Cylindrobasidium ipidophilum (Physalacriaceae). The free-living wood-decay fungus Phlebiopsis gigantea (Phanerochaetaceae) has been occasionally associated with bark beetles but is unrelated to C. ipidophilum or Entomocorticium.

Detection of Colletotrichum acutatum Sensu Lato on Strawberry by Loop-Mediated Isothermal Amplification.

Colletotrichum acutatum, one of the most economically damaging pathogens of strawberry, is the primary causal agent of anthracnose fruit rot (AFR). A key challenge in managing AFR is detecting the pathogen on asymptomatic plants. To meet this need, a loop-mediated isothermal amplification (LAMP) assay was developed that incorporated two sets of primers: LITSG1, targeted on the intergenic transcribed spacer (ITS) region of ribosomal DNA, and Ltub2, on the ß-tubulin 2 gene. In pure culture assays, Ltub2 was specific for detection of C. acutatum, whereas LITSG1 detected C. acutatum and two additional anthracnose pathogens, C. gloeosporioides and C. fragariae. LITSG1 had 10-fold lower detection threshold (20 pg of mycelial DNA) than Ltub2 (200 pg mycelial DNA) in detection of C. acutatum from pure culture. For detection on asymptomatic leaves, two protocols for dislodging C. acutatum for DNA extraction were compared: i) the sonicate-agitate (SA) method and ii) the freeze-incubate-sonicate-agitate (FISA) method, which initially freezes tissues, followed by 2 days of incubation at 26°C in darkness, and then, sonication and agitation. Both methods were used for greenhouse-grown plant leaves that had been spray inoculated with serial dilutions ranging from 1.5 × 106 to 1.5 conidia ml-1. The FISA method produced more repeatable results than the SA method. For the FISA method, detection limits (expressed as initial inoculum concentrations) using LITSG1 and Ltub2 were 1.5 × 101 and 1.5 × 102 conidia ml-1, respectively. For composite samples comprised of inoculated (1.5 × 106 conidia ml-1) and noninoculated leaves of greenhouse-grown strawberry, the two sets of LAMP primers were compared using the SA method. Primer set LITSG1 consistently detected the pathogen from a single inoculated leaf in bulk samples of 50 or fewer pathogen-free leaves, whereas Ltub2 consistently detected one inoculated leaf in 20 or fewer pathogen-free leaves. Using primer set LITSG1, FISA was more sensitive than SA for detecting C. acutatum on leaves of field-grown plants from Florida. In an Iowa field trial using the FISA method, both primer sets detected C. acutatum in samples of asymptomatic leaves 6 days before fruit symptoms appeared. The results indicate that the LAMP assay has potential to provide a simplified method for detection of C. acutatum on asymptomatic strawberry plants.

Phenology of Infection on Apple Fruit by Sooty Blotch and Flyspeck Species in Iowa Apple Orchards.

Sooty blotch and flyspeck (SBFS) is a fungal disease complex that can cause significant economic losses to apple growers by blemishing the fruit surface with dark-colored colonies. Little is known about the phenology of host infection for this diverse group of epiphytes. In 2009 and 2010, we investigated the timing of infection of apple fruit by SBFS species in six commercial apple orchards in Iowa. Five trees in each orchard received no fungicide sprays after fruit set. Within 3 weeks after fruit set, 60 apples per tree were covered with Japanese fruit bags to minimize inoculum deposition. Subsequently, a subsample of bagged apples was exposed for a single 2-week-long period and then rebagged for the remainder of the growing season. Experimental treatments included seven consecutive 2-week-long exposure periods; control treatments were apples that were either bagged or exposed for the entire season. After apples had been stored at 2°C for 6 weeks following harvest, all SBFS colonies on the apples were identified to species using a PCR-RFLP protocol. A total of 15 species were identified. For the seven most prevalent species, the number of infections per cm2 of fruit surface was greatest on apples that had been exposed early in the season. Two SBFS species, Peltaster fructicola and Colletogloeopsis-like FG2, differed significantly from each other in time required to attain 50% of the total number of colonies per apple, and analysis of variance indicated a significant interaction of SBFS taxon with exposure period. Our findings are the first evidence of species-specific patterns in timing of SBFS inoculum deposition and infection on apple fruit, and strengthen previous observations that most SBFS infections resulting in visible colonies at harvest develop from infections that occur early in the fruit development period. By defining taxon-specific phenological patterns of fruit infection, our findings, when combined with knowledge of region-specific patterns of taxon prevalence, provide a foundation for development of more efficient and cost-effective SBFS management tactics.

Genetic and virulence variability among Erwinia tracheiphila strains recovered from different cucurbit hosts.

The causal agent of cucurbit bacterial wilt, Erwinia tracheiphila, has a wide host range in the family Cucurbitaceae, including economically important crops such as muskmelon (Cucumis melo), cucumber (C. sativus), and squash (Cucurbita spp.). Genetic variability of 69 E. tracheiphila strains was investigated by repetitive-element polymerase chain reaction (rep-PCR) using BOXA1R and ERIC1-2 primers. Fingerprint profiles revealed significant variability associated with crop host; strains isolated from Cucumis spp. were clearly distinguishable from Cucurbita spp.-isolated strains regardless of geographic origin. Twelve E. tracheiphila strains isolated from muskmelon, cucumber, or summer squash were inoculated onto muskmelon and summer squash seedlings, followed by incubation in a growth chamber. Wilt symptoms were assessed over 3 weeks, strains were reisolated, and rep-PCR profiles were compared with the inoculated strains. Wilting occurred significantly faster when seedlings were inoculated with strains that originated from the same crop host genus (P<0.001). In the first run of the experiment, cucumber and muskmelon strains caused wilting on muskmelon seedlings at a median of 7.8 and 5.6 days after inoculation (dai), respectively. Summer squash seedlings wilted 18.0, 15.7, and 5.7 dai when inoculated with muskmelon-, cucumber-, and squash-origin strains, respectively. In a second run of the experiment, cucumber and muskmelon strains caused wilting on muskmelon at 7.0 and 6.9 dai, respectively, whereas summer squash seedlings wilted at 23.6, 29.0 and 9.0 dai when inoculated with muskmelon-, cucumber-, and squash-origin strains, respectively. Our results provide the first evidence of genetic diversity within E. tracheiphila and suggest that strain specificity is associated with plant host. This advance is a first step toward understanding the genetic and population structure of E. tracheiphila.

Dissoconiaceae associated with sooty blotch and flyspeck on fruits in China and the United States.

Zasmidium angulare, a novel species of Mycosphaerellaceae, and several novel taxa that reside in Dissoconiaceae, were identified from a collection of apples and Cucurbita maxima (cv. Blue Hubbard) from China and the USA that exhibited sooty blotch and flyspeck (SBFS) signs on their host substrata. Morphology on fruit surfaces and in culture, and phylogenetic analyses of the nuclear ribosomal DNAs 28S and internal transcribed spacer regions, as well as partial translation elongation factor 1-alpha gene sequences in some cases, were used to delineate seven previously unidentified species and three known species. Pseudoveronaea was established as a new genus of Dissoconiaceae, represented by two species, P. ellipsoidea and P. obclavata. Although Pseudoveronaea was morphologically similar to Veronaea, these fungi clustered with Dissoconiaceae (Capnodiales) rather than Chaetothyriales (Herpotrichiellaceae). Ramichloridium mali comb. nov., and three novel species, R. cucurbitae, R. luteum and R. punctatum were closely related with R. apiculatum, which together formed a distinct subclade in Dissoconiaceae. Species of Dissoconium s.lat. clustered in two well-supported clades supported by distinct morphological and cultural features. Subsequently Uwebraunia, a former synonym of Dissoconium, was resurrected for the one clade, with new combinations proposed for U. australiensis, U. commune, U. dekkeri and U. musae. Furthermore, we also reported that D. aciculare, Dissoconium sp., U. commune and U. dekkeri were associated with SBFS on apples.

Temporal patterns in appearance of sooty blotch and flyspeck fungi on apples.

Sooty blotch and flyspeck (SBFS) is a complex of about 80 fungal species that blemish the surface of apple fruit in humid regions worldwide. The dark colonies become visible in mid- to late summer, reducing the value of fresh fruit. Although many SBFS species can co-occur in the same orchard and even on the same apple, little is known about temporal patterns of these species, including the timing of colony appearance. To test the hypothesis that colonies of SBFS species appear on apples at characteristic times during the growing season, 50 apples were monitored weekly at three Iowa orchards in 2006 and six orchards in 2007 and 2008. However, a mean of 24.3 apples per orchard was assessed at harvest because of apple drop throughout the season. Colonies were marked with colored pens as they appeared. After harvest and after storage of apples at 2 °C for 3 months, SBFS colonies on each fruit were counted and classified by morphology, and a representative subset of colonies was excised from the fruit and preserved on dried peels for species identification using rDNA. Seventeen species were identified. Stomiopeltis spp. RS1 and RS2 appeared on apples 10 to 14 days before other SBFS taxa. Dissoconium aciculare was generally the last species to appear on apple fruit, and it continued to appear during postharvest storage. The most prevalent taxa in Iowa orchards were also the most abundant. Diversity of SBFS fungi in an orchard was positively correlated with cumulative hours of surface wetness hours due to rainfall or dew, which is believed to favor growth of SBFS fungi. Species-specific information about temporal patterns of appearance on apple fruit may lead to improved SBFS management strategies.

Feasibility of Delaying Removal of Row Covers to Suppress Bacterial Wilt of Muskmelon (Cucumis melo).

Bacterial wilt, caused by Erwinia tracheiphila, is a major disease of cucurbit crops in the United States. Management of the disease relies on controlling two vector species, striped (Acalymma vittatum) and spotted (Diabrotica undecimpunctata) cucumber beetles. Six field trials were conducted at Iowa State University research farms during 2007, 2008, and 2009 to assess the efficacy of delayed removal of spunbond polypropylene row covers to control bacterial wilt on muskmelon (Cucumis melo). Treatments were (i) row cover removed at anthesis (conventional timing of removal), (ii) covers removed 10 days after row cover ends were opened at anthesis, (iii) covers removed 10 days after bumble bee hives were inserted under row covers at anthesis, and (iv) a noncovered control. In two field trials during 2007 and 2008, the delayed-removal row-cover treatments significantly suppressed bacterial wilt throughout the growing season and enhanced yield compared with the noncovered and removal-at-anthesis controls. In Gilbert in 2008, however, bacterial wilt suppression was equivalent among all three row-cover treatments. No bacterial wilt was observed during three trials in 2009, and there was minimal difference in marketable yield among treatments. Net returns were compared using partial budget and sensitivity analyses. Melon prices and occurrence of bacterial wilt had a strong impact on net returns. Using row covers increased production costs by 45%. In site years in which bacterial wilt occurred, delaying removal of row covers resulted in the highest returns. When bacterial wilt was absent, however, the delayed-removal row-cover treatments had the lowest returns. Results of the sensitivity analysis indicated that delaying removal of row covers for 10 days could be a cost-effective component of an integrated bacterial wilt suppression strategy for muskmelon where bacterial wilt occurs ≥50% of production seasons.

Novel fungal genera and species associated with the sooty blotch and flyspeck complex on apple in China and the USA.

Fungi in the sooty blotch and flyspeck (SBFS) complex cause blemishes on apple and pear fruit that result in economic losses for growers. The SBFS fungi colonise the epicuticular wax layer of pomaceous fruit but do not invade the cuticle. Fungi causing fuliginous and punctate mycelial types on apple are particularly difficult to identify based on morphological criteria because many species in the SBFS complex share the same mycelial phenotypes. We compared the morphology and nuclear ribosomal DNA phylogeny (ITS, LSU) of 11 fungal strains isolated from SBFS blemishes on apple obtained from two provinces in China and five states in the USA. Parsimony analysis, supported by cultural characteristics and morphology in vitro, provided support to delimit the isolates into three novel genera, representing five new species. Phaeothecoidiella, with two species, P. missouriensis and P. illinoisensis, is introduced as a new genus with pigmented endoconidia in the Dothideomycetes. Houjia (Capnodiales) is introduced for H. pomigena and H. yanglingensis. Although morphologically similar to Stanjehughesia (Chaetosphaeriaceae), Houjia is distinct in having solitary conidiogenous cells. Sporidesmajora (Capnodiales), based on S. pennsylvaniensis, is distinguished from Sporidesmium (Sordariomycetes) in having long, multiseptate conidiophores that frequently have a subconical, darkly pigmented apical cell, and very long, multi-euseptate conidia.

First Report of Seven Species of Sooty Blotch and Flyspeck Fungi on Asimina triloba in Iowa.

Fungi in the sooty blotch and flyspeck (SBFS) complex cause major economic losses on cultivated pome fruits in humid regions worldwide and also colonize many species of reservoir host plants. In 2007, 10 mature fruit of pawpaw (Asimina triloba), a native tree in eastern North America exhibiting SBFS colonies on the epicuticular wax layer, were collected from wild trees in eastern Iowa. Colonies of SBFS fungi on the fruit were described according to mycelial type (1). Isolates of representative colonies on acidified water agar were subcultured on potato dextrose agar and the morphological characters were observed. After DNA was extracted from cultures and amplified by PCR using primer set ITS-1F/ITS4, 470-bp sequences were compared with those of previously identified SBFS species using NCBI BLAST. The BLAST analysis showed 100% homology of the sequences with six species that had been previously confirmed to cause SBFS on apple fruit by fulfilling Koch's postulates (1): Colletogloeum sp. FG2, Dissoconium aciculare, Peltaster sp. P2.1, P. fructicola, Stomiopeltis versicola, and Stomiopeltis sp. RS1 (GenBank Accession Nos. AY598907, AY598874, AY5988888, AY598887, AY5160165, and AY598882, respectively). Using the NCBI bl2seq application, Dothideomycete sp. CS2, an additional previously confirmed SBFS species, was revealed by sequence homology. Morphology of some SBFS species on pawpaw differed from that on apple. For example, Colletogloeum sp. FG2, which produces the fuliginous mycelial type (1) on apple fruit, developed the ridged honeycomb mycelial type on pawpaw fruit. D. aciculare and Stomiopeltis sp. RS1 produced the compact speck mycelial type on pawpaw, but are known to develop discrete speck and ramose mycelial types, respectively, on apple. These differences may result from host species differences in the epicuticular wax layer of the fruit. To our knowledge, this is the first report of SBFS fungi on A. triloba in North America, although the SBFS species Zygophiala jamaicensis was reported on the same host in Japan (2). Identifying SBFS fungi on reservoir host plants is an important step toward improving disease management strategies. References: (1) J. Batzer et al. Mycologia 97:1268, 2005. (2) H. Nasu and H. Kunoh Plant Dis. 71:361, 1987.

An RFLP-Based Technique for Identifying Fungi in the Sooty Blotch and Flyspeck Complex on Apple.

A restriction fragment length polymorphism (RFLP)-based technique was developed to identify members of the sooty blotch and flyspeck (SBFS) disease complex on apple because these fungi are difficult to identify using agar-plate isolation and morphological description. The method includes polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) using a fungal-specific forward primer (ITS1-F) and an SBFS-specific reverse primer (Myc1-R), followed by digestion of the PCR product by the HaeIII restriction enzyme. When applied to previously identified isolates of 24 SBFS-causing species in nine genera, the PCR-RFLP assay produced 14 unique banding patterns. Different genera never shared the same RFLP pattern. To evaluate performance in vivo, the technique was applied to DNA extracted directly from SBFS colonies on apple fruit from three Iowa orchards. The primers amplified the rDNA of only SBFS fungi, with the exception of a Cladosporium sp.; however, its RFLP banding pattern was distinct from those of SBFS fungi. The majority (60%) of SBFS colonies in the in vivo trial were identified to genus by RFLP analysis. The PCR-RFLP assay greatly streamlined the identification process by minimizing the need for culturing, indicating its value as a tool for field studies of the SBFS complex.

Spatial Heterogeneity of Leaf Wetness Duration in Apple Trees and Its Influence on Performance of a Warning System for Sooty Blotch and Flyspeck.

To determine the effect of sensor placement on the performance of a disease-warning system for sooty blotch and flyspeck (SBFS), we measured leaf wetness duration (LWD) at 12 canopy positions in apple trees, then simulated operation of the disease-warning system using LWD measurements from different parts of the canopy. LWD sensors were placed in four trees within one Iowa orchard during two growing seasons, and in one tree in each of four orchards during a single growing season. The LWD measurements revealed substantial heterogeneity among sensor locations. In all data sets, the upper, eastern portion of the canopy had the longest mean daily LWD, and was the first site to form dew and the last to dry. The lower, western portion of the canopy averaged about 3 h less LWD per day than the top of the canopy, and was the last zone where dew formed and the first to dry off. On about 25% of nights when dew occurred in the top of the canopy, no dew formed in the lower, western canopy. Intracanopy variability of LWD was more pronounced when dew was the sole source of wetness than on days when rainfall occurred. Daily LWD in the upper, eastern portion of the canopy was slightly less than reference measurements made at a 0.7-m height over turfgrass located near the orchard. When LWD measurements from several canopy positions were input to the SBFS warning system, timing of occurrence of a fungicide-spray threshold varied by as much as 30 days among canopy positions. Under Iowa conditions, placement of an LWD sensor at an unobstructed site over turfgrass was a fairly accurate surrogate for the wettest part of the canopy. Therefore, such an extra-canopy LWD sensor might be substituted for a within-canopy sensor to enhance operational reliability of the SBFS warning system.

First Report of Five Sooty Blotch and Flyspeck Fungi on Prunus americana in the United States.

The sooty blotch and flyspeck (SBFS) complex includes more than 30 fungi that blemish the cuticle of apple fruit, causing economic losses in humid regions worldwide (1). In August 2005, we sampled SBFS-infested wild plum (Prunus americana) fruit growing in hedgerows in Iowa. Colonies were categorized according to mycelial type (1), and isolates were made from representative colonies onto acidified water agar (AWA). Plum skins with SBFS signs were excised, pressed, and photographed. DNA was obtained from purified isolates and also from mycelium and fruiting bodies scraped directly from plum fruit skins. Extracted DNA was amplified using primer pair ITS1-F/Myc1-R (ACTCGTCGAAGGAGCTACG) and PCR products were sequenced using primer pair ITS-1F/ITS4. Six sequences were obtained from pure cultures and seven from colonies on plum fruit skin. BLAST analysis of the 470-bp sequences showed 100% homology to five known species in the SBFS complex: Zygophiala cryptogama, Zygophiala wisconsinensis, Pseudocercosporella sp. RH1, and Stomiopeltis spp. RS1 and RS2 (GenBank Accession Nos. AY598854, AY598853, AY5988645, AY598882, and AY598883, respectively). Observations of colony and fruiting structure morphology from cultures on potato dextrose agar (PDA) and colonies on plums confirmed species identity. A modified version of Koch's postulates was conducted to verify that these fungi caused the signs observed on plum and could also infest apple fruit. In June 2006, 1-month-old cultures on PDA were pulverized in a blender with sterile distilled water, passed through four layers of sterile cheesecloth, and transferred to sterile jars. Each isolate was inoculated onto 20 fruit on plum trees (P. americana) on the Iowa State University (ISU) campus and 20 fruit on cv. Golden Delicious apple trees at the ISU Research Station, Gilbert, IA. Each fruit was disinfested with 70% ethanol, air dried, swabbed with inoculum, and covered with a Fuji bag. At harvest, fungal colonies on fruit were reisolated onto AWA. DNA was extracted from pure cultures; when isolations on agar were unsuccessful, DNA was extracted directly from colonies on fruit. PCR was conducted using ITS1-F/Myc1-R, and PCR products were sequenced using ITS1-F/ITS4. All five species were reisolated and sequenced from apple. Pseudocercosporella sp. RH1 and Stomiopeltis sp. RS1 were sequenced from inoculated plums. Although flyspeck, presumably caused by Schizothyrium pomi, was reported on Japanese plum (P. salicina) in Japan (2) and black cherry (P. serotina) in the United States (3), to our knowledge this is the first report of SBFS fungi on plum in the United States and the first confirmation that fungi from plum can produce SBFS signs on apple fruit. Wild plum may therefore act as a reservoir host, providing inoculum for SBFS infestations on apple. References: (1) J. Batzer et al. Mycologia 97:1268, 2005. (2) H. Nasu and H. Kunoh. Plant Dis. 71:361, 1987. (3) T. B. Sutton. Plant Dis. 72:801, 1988.

Evaluation of Postharvest Removal of Sooty Blotch and Flyspeck on Apples Using Sodium Hypochlorite, Hydrogen Peroxide with Peroxyacetic Acid, and Soap.

Postharvest dips of apples (Malus × domestica) in commercial disinfestants were used to remove signs of the flyspeck (FS) pathogen, Schizothyrium pomi, and the sooty blotch (SB) complex (Peltaster fructicola, Leptodontium elatius, and Geastrumia polystigmatis). Apples were dipped for 7 or 15 min in buffered sodium hypochlorite (Agclor 310 plus Decco 312 Buffer) at 200, 400, 500, 600, or 800 ppm chlorine, a mixture of hydrogen peroxide and peroxyacetic acid (Tsunami 100) at 60 ppm/80 ppm, 120 ppm/160 ppm, or 360 ppm/480 ppm, respectively, or soap (Kleen 440), then brushed and rinsed for 30 s on a commercial grading line. Disease severity was assessed as percent diseased area using a quantitative rating system, and by counting the number of colonies of three mycelial types of SB and FS. Percent diseased area on apples was converted to USDA apple grade ratings and retail values. Both assessment methods provided similar results, but the percent-diseased-area method was less labor intensive. A 7-min dip in 800 ppm chlorine resulted in a mean increase from 25 and 55% to 100% Extra Fancy grade for 'Jonathan' and 'Golden Delicious' apples, respectively, and increased market value by 31 and 14%, respectively. The 7-min, 200-ppm chlorine dip resulted in an increase from 28 and 45% to 92.5 and 96.5% Extra Fancy after treatment for 'Jonathan' and 'Golden Delicious', respectively. Blemishes were removed more effectively from 'Jonathan' and 'McIntosh' apples than from 'Golden Delicious'. Mycelial types of the sooty blotch and flyspeck fungi were removed differentially by the disinfestant dip treatments.

Responses of 11 Fraxinus Cultivars to Ash Yellows Phytoplasma Strains of Differing Aggressiveness.

Five cultivars of Fraxinus americana (white ash) and five of F. pennsylvanica (green ash) were graft-inoculated with three strains of ash yellows phytoplasmas at Ames, IA, and with thrsee other strains at Ithaca, NY. A sixth green ash cultivar was tested only in New York. Trees were allowed to grow in field plots for 3 years. Infection was detected via the DAPI (4', 6-diamidino-2-phenylindole 2HCl) fluorescence test. Incidence of witches'-brooms on infected trees was greater on white ash than green ash and varied significantly among phytoplasma strain treatments at both locations. Volume growth of infected ash, averaged across cultivars over 2 years in Iowa and 3 years in New York, was 49 and 59%, respectively, as great as that of noninfected trees. Foliar greenness was reduced significantly by infection at both locations, and this reduction was positively correlated with growth reduction. Cultivars at each location varied significantly in growth of noninfected trees and in growth of diseased trees relative to that of nonin-fected trees (a measure of phytoplasma tolerance), but cultivar means for these variables in Iowa were not significantly correlated with those in New York. Green ash cvs. Bergeson, Dakota Centennial, and Patmore and white ash cv. Autumn Applause were above average in tolerance at both locations. Phytoplasma strains at each location varied significantly in aggressiveness as indicated by host growth suppression.