Pisolithus Species Research Articles

Speciation Underpinned by Unexpected Molecular Diversity in the Mycorrhizal Fungal Genus Pisolithus.

The mutualistic ectomycorrhizal (ECM) fungal genus Pisolithus comprises 19 species defined to date which colonize the roots of >50 hosts worldwide suggesting that substantial genomic and functional evolution occurred during speciation. To better understand this intra-genus variation, we undertook a comparative multi-omic study of nine Pisolithus species sampled from North America, South America, Asia, and Australasia. We found that there was a small core set of genes common to all species (13%), and that these genes were more likely to be significantly regulated during symbiosis with a host than accessory or species-specific genes. Thus, the genetic "toolbox" foundational to the symbiotic lifestyle in this genus is small. Transposable elements were located significantly closer to gene classes including effector-like small secreted proteins (SSPs). Poorly conserved SSPs were more likely to be induced by symbiosis, suggesting that they may be a class of protein that tune host specificity. The Pisolithus gene repertoire is characterized by divergent CAZyme profiles when compared with other fungi, both symbiotic and saprotrophic. This was driven by differences in enzymes associated with symbiotic sugar processing, although metabolomic analysis suggest that neither copy number nor expression of these genes is sufficient to predict sugar capture from a host plant or its metabolism in fungal hyphae. Our results demonstrate that intra-genus genomic and functional diversity within ECM fungi is greater than previously thought, underlining the importance of continued comparative studies within the fungal tree of life to refine our focus on pathways and evolutionary processes foundational to this symbiotic lifestyle.

Evolutionary Insights Into Two Widespread Ectomycorrhizal Fungi (Pisolithus) From Comparative Analysis of Mitochondrial Genomes.

The genus Pisolithus is a group of global ectomycorrhizal fungi. The characterizations of Pisolithus mitochondrial genomes have still been unknown. In the present study, the complete mitogenomes of two Pisolithus species, Pisolithus microcarpus, and Pisolithus tinctorius, were assembled and compared with other Boletales mitogenomes. Both Pisolithus mitogenomes comprised circular DNA molecules with sizes of 43,990 and 44,054 bp, respectively. Comparative mitogenomic analysis showed that the rps3 gene differentiated greatly between Boletales species, and this gene may be subjected to strong pressure of positive selection between some Boletales species. Several plasmid-derived genes and genes with unknown functions were detected in the two Pisolithus mitogenomes, which needs further analysis. The two Pisolithus species show a high degree of collinearity, which may represent the gene arrangement of the ancestors of ectomycorrhizal Boletales species. Frequent intron loss/gain events were detected in Boletales and basidiomycetes, and intron P717 was only detected in P. tinctorius out of the eight Boletales mitogenomes tested. We reconstructed phylogeny of 79 basidiomycetes based on combined mitochondrial gene dataset, and obtained well-supported phylogenetic topologies. This study served as the first report on the mitogenomes of the family Pisolithaceae, which will promote the understanding of the evolution of Pisolithus species.

Two new species of Pisolithus (Sclerodermataceae) from Australasia, and an assessment of the confused nomenclature of P. tinctorius

Based on morphological data and ITS nrDNA sequence analyses, we formally describe two species from the phylogenetic grouping ‘Martin sp. 10’ as Pisolithus thermaeus sp. nov., endemic to New Zealand, and P. tympanobaculus sp. nov. from Australia. All Pisolithus species in New Zealand are restricted in distribution to geothermally active areas, in ectomycorrhizal association with Kunzea tenuicaulis. In Australia species occur in a broad range of habitats in association with Eucalyptus or Acacia species; and in New Caledonia species form associations with various Myrtaceae or an Acacia species. The distribution ranges of Pisolithus albus, P. marmoratus and P. croceorrhizus are extended to include the Kimberley and Top End regions of northern Australia. We also confirm that neither P. arenarius, nor P. tinctorius sensu stricto, nor P. arhizus are currently known to occur in Australia or New Zealand. The long-used but never formalized combination Pisolithus albus (Cooke & Massee) Priest is validated here and nomenclatural knots relating to three different usages of the name Pisolithus tinctorius are unraveled and suggestions made for their resolution.

Intraspecific variability of Pisolithus spp. as a response to changes in soil characteristics in a Moroccan cork oak plantation

Soil characteristics may be an important factor in structuring fungal communities. We relate the above- and below-ground distribution of the ectomycorrhizal fungi (EMF) Pisolithus spp. to changes in soil chemistry in a Quercus suber plantation located in the Maâmora forest (Morocco). Intrageneric variability of 115 fruit bodies was studied, using morphological characterization, restriction fragment length polymorphism (RFLP) analyses and internal transcribed spacer (ITS) sequencing. Two Pisolithus spp. genotypes were identified: 97% of the fruit bodies represent Pisolithus arhizus; the remaining 3% correspond to Pisolithus species 4, as previously described by Martin et al. [New Phytologist 153 (2002) 345-357]. Multivariate analysis (PCA) showed that species structure was strongly correlated with soil characteristics. P. arhizus fruit bodies were frequently found in the Eastern part of the plot at low molasic clay, N and P contents, while Pisolithus species 4 were collected only in the Western part at the high molasic clay, N and P contents. To determine whether this change in fruit body structure was expressed at the belowground level, we used morphological and molecular techniques to characterize the Pisolithus ectomycorrhizas. Only ectomycorrhizas of P. arhizus were identified at the low P–N–molasic clay site and disappeared completely at the high P–N–red clay site, where no Pisolithus species 4 mycorrhizas were found. However, autocorrelation among soil parameters makes it difficult to isolate the effects of individual parameters. These results show the local-scale impact of natural spatial heterogeneity on an ectomycorrhizal fungal genus.

Molecular phylogeny of Pisolithus species from Moroccan forest woodlands

The phylogenetic relationships among 200 Pisolithus basidiomata collected from pine, oak, and eucalypt forests and rockrose scrubs in Morocco were investigated. Using PCR-RFLP analysis of the internal transcribed spacer (ITS) of ribosomal DNA, this collection could be divided into 5 groups, by using PCR-RFLP analysis of the internal transcribed spacer (ITS) of ribosomal DNA. The ITS of a representative basidioma of each group was sequenced, and a phylogenetic analysis was performed. The dendrogram suggests that at least five Pisolithus species are present in Morocco. Pisolithus basidiomata collected in the Pinus and Quercus forests correspond to Pisolithus arrhizus and P. species 4 as previously described by Martin and colleagues in 2002. Those collected from the eucalyptus forests, under E. gomphocephala and E. camaldulensis, correspond to P. albus and P. microcarpus. Basidiomata collected from the rockrose scrubs, under Cistus crispus, C. monspeliensis or C. salviifolius, are all identified as Pisolithus species 3. Phylogenetic analyses showed that our different Pisolithus grouped well with Pisolithus specimens from other geographical origins.

Effects of sodium chloride on growth of ectomycorrhizal fungal isolates in culture

We studied the tolerance of ectomycorrhizal (ECM) fungi to sodium chloride (NaCl) to find the best fungus to aid growth of Pinus thunbergii. Four ECM fungi, Cenococcum geophilum, Pisolithus tinctorius, Rhizopogon rubescens, and Suillus luteus, were grown in liquid MMN media with five different concentrations of NaCl for 30 days, and their mycelial weights were determined. Mycelial weights of P. tinctorius and R. rubescens were not significantly different between 0 mM and 200 mM, whereas those of C. geophilum and S. luteus decreased with increasing NaCl concentration, indicating that the former two species were more tolerant to higher NaCl concentrations than the latter species. We further studied the intraspecific differences in NaCl tolerance of nine P. tinctorius isolates. They were grown on MMN agar media with six different concentrations of NaCl for 21 days, and their radial growth was measured. In total, the hyphal growth at 25 mM NaCl was significantly higher than those at the other NaCl concentrations, and EC50 values were confirmed at between 50 mM and 200 mM. Among the isolates, Pt03 and Pt21 showed measurable growth at 200 mM; the growth of Pt03 was not significantly different between 0 mM and 200 mM. The results indicate that there are intraspecific variations in NaCl tolerance of Pisolithus species.

Abundance and characteristics of Pisolithus ectomycorrhizas in New Zealand geothermal areas

Pisolithus is restricted in New Zealand to geothermal areas where it associates with Kunzea ericoides var. microflora (prostrate kanuka) and occasionally Leptospermum scoparium. Here we describe for the first time the ectomycorrhizal morphotypes of three New Zealand Pisolithus species and report the frequency and abundance of these morphotypes against other mycorrhizal fungi associated with these hosts in New Zealand geothermal areas. The three Pisolithus species form typical ectomycorrhizal associations with Kunzea ericoides var. microflora, and one also was observed forming typical ectomycorrhizal associations with Leptospermum scoparium. Although the morphotypes from the three Pisolithus species share many morphological and anatomical characteristics, they vary with regard to the abundance of rhizomorphs. The common occurrence of Pisolithus fruiting bodies at the geothermal sites was matched by frequent and abundant Pisolithus ectomycorrhizas. Pisolithus ectomycorrhizas were frequent (100% of soil cores) and abundant (between 55 and 88% of ectomycorrhizal tips) associates of prostrate kanuka in hot (50 C at 8 cm depth), highly acidic and N depleted soils. The levels of arbuscular mycorrhizal colonization of prostrate kanuka were lower than on K. ericoides and L. scoparium on cooler soils. The stressful conditions where prostrate kanuka dominates probably favor Pisolithus over the mycorrhizal fungi occurring in cooler geothermal areas. Questions about how several genetically similar Pisolithus species co-occur on prostrate kanuka in geothermal areas without mutual competitive exclusion are discussed.

Genetic diversity of Pisolithus in New Zealand indicates multiple long-distance dispersal from Australia.

• Pisolithus is a common ectomycorrhizal (EcM) associate of prostrate kanuka Kunzea ericoides var. microflora (Myrtaceae) in New Zealand geothermal areas. Here, we report the genetic diversity and phylogeny of Pisolithus and interpret the results in relation to the origin of this fungus in New Zealand. • We determined the genetic variation of Pisolithus on the basis of ITS gene sequences and spore morphology. • We identified three Pisolithus species in New Zealand, each matching Australian species associated with eucalypts and acacias. All three species co-occurred locally in thermal areas, with two species sometimes colonizing root tips in the same soil volume, indicating co-occurrence of species on a smaller scale. • We propose that Pisolithus fungi were introduced to New Zealand from Australia by trans-Tasman airflow during recent geological times. The success of this long-distance dispersal of EcM fungi may be related to the capacity of kanuka to act as a 'nurse plant' for wind-blown spores.

Molecular phylogeny of ectomycorrhizal Pisolithus fungi associated with pine, dipterocarp, and eucalyptus trees in Thailand

The phylogenetic relationships among 135 Pisolithus basidiomes and two isolates collected from three pine forests, a pine-dipterocarp forest, two dipterocarp forests, and 29 eucalyptus plantations in Thailand were investigated. Internal transcribed spacer (ITS) polymorphism analyses, including terminal RFLP, divided them into 26 groups. The ITS in a representative basidiome of each group was sequenced, and a phylogenetic analysis was performed. The dendrogram suggested that at least three Pisolithus species are present in Thailand. Pisolithus basidiomes collected in the pine forests and under some Shorea roxburghii trees in a pine-dipterocarp forest corresponded to species 5 as previously described by Martin et al. in 2002. Those collected under S. roxburghii and Dipterocarp alatus trees in the dipterocarp forests did not match any previously reported species. Basidiomes collected from the eucalyptus plantations were all identified as Pisolithus albus.

Persistence of some Australian Pisolithus species introduced into eucalypt plantations in China

Some eucalypt plantations in south China grow poorly because of soil infertility and low diversity of compatible ectomycorrhizal fungi. One option to improve productivity of plantations is to introduce beneficial fungi. In order to evaluate persistence of introduced symbiotic fungi, there is a need to discriminate them from any indigenous related species. Eucalypt mycorrhizal trial sites, established for nutrition and survival studies, were used for this purpose. Seedlings of Eucalyptus camaldulensis and Eucalyptus urophylla had been inoculated with pure cultures of selected Australian ectomycorrhizal fungi, including three isolates of Pisolithus albus and one isolate each of two unnamed Pisolithus spp., and ectomycorrhizal seedlings had been out-planted at two sites in Guangzhou Province, People’s Republic of China. Sequences of the internal transcribed spacers (ITSs) of ribosomal DNA (rDNA) from inoculant fungi and fungi re-isolated from sporocarps in the field were compared. Sequencing confirmed that an Australian isolate (H4111) of one unnamed Pisolithus spp. had persisted in competition with an indigenous Pisolithus spp. Selected isolates of Australian Pisolithus formed macro- and micro-morphologically typical ectomycorrhizas in pure culture syntheses and in the field. In comparison, the indigenous Chinese Pisolithus formed an incomplete association with a poorly developed mantle. Comparisons between ITS sequences from Pisolithus isolates associated with Eucalyptus spp. from elsewhere in the world and ITS sequences of Australian Pisolithus spp. indicate that the same unnamed Pisolithus spp. has also become established in Portugal, Brazil and South Africa. Because Pisolithus isolate H4111 produced sporocarps in south China under eucalypts and promoted tree growth, this fungus would be useful in spore orchards to provide spore inoculum for eucalypt nurseries. The local Chinese Pisolithus is not recommended for inoculation programs because it is ineffective in forming mycorrhizas with eucalypts. The isolate H4111 is from a Pisolithus that occurs naturally along the east coast of Australia. Although this Pisolithus has been widely dispersed with eucalypts in other parts of the world, it is not present in eucalypt plantations in east Asia.

Dynamics of ectomycorrhizal fungi in an Eucalyptus globulus plantation: effect of phosphorus fertilization

Eucalyptus globulus is the most widely planted temperate eucalypt in regions of Mediterranean climate. Plantations in Western Australia (WA) are common on sandy soils where fertilization is necessary (especially N, P, K and Cu) to sustain high growth rates. Ectomycorrhizal (ECM) fungi are common in these plantations and are probably ecologically important in nutrient cycling. Whether fertilization with phosphorus (P) could alter fungal dynamics in these plantations is investigated in this study. Three rates of P (nil, low=100 kg P ha −1 and high=1000 kg P ha −1 ), including basal nutrients, were applied to a 4-years-old E. globulus plantation in WA and the following measured over a 2 years period: fungal diversity, fungal biomass, soil hyphal production, tree biomass, litter production, and accumulation of N, P, K and Cu in the fungal and tree components. The addition of P reduced the number of basidiocarps by 5 (low P)–14% (high P), basidiocarp and soil hyphal biomass by 26–40% and 29–44%, respectively, without affecting above-ground ECM fungal diversity, species of Laccaria, Pisolithus and Scleroderma. There was no response in whole tree biomass or litterfall to P, but low P increased fine root biomass in ingrowth cores at 6 months to 364 kg ha −1. Accumulation of N, P, K and Cu generally declined in fungi and litterfall with P, but increased in tree biomass (except N and Cu). The total nutrient load was in the order: above- ground tree > soil hyphae > below- ground tree > litter> fine root > basidiocarp . The combined nutrient pool in the fungal component was approximately 10% of that in the tree. This illustrates the potential significance of ECM fungi in immobilizing P and other nutrients, and in their subsequent cycling during fungal decomposition in plantations. The impact of P on fungal dynamics therefore requires consideration when applying fertilizers in eucalypt plantations.

Phylogeography of the ectomycorrhizal Pisolithus species as inferred from nuclear ribosomal DNA ITS sequences

Summary The fungal genus Pisolithus is cosmopolitan in warm temperate regions and forms ectomycorrhizal associations with a wide range of woody plants. To delimit phylogenetic Pisolithus species and identify their geographical distribution, 102 collections were made worldwide and their rDNA internal transcribed spacer (ITS) was sequenced. Phylogenetic analyses of these sequences, together with 46 additional GenBank accessions, identified 11 species. A strong phylogeographical pattern was observed related to the native geographical origin of the host plants. In the Holarctic, P. tinctorius was widely distributed, associated with Pinus and Quercus. It has been co‐introduced with pines to other biogeographical regions. Several Pisolithus lineages, including P. aurantioscabrosus, occurred in restricted biogeographical regions associated with endemic plants, such as Afzelia in eastern Africa. Pisolithus albus, P. marmoratus and P. microcarpus were associated with Australasian hosts (Eucalyptus, Acacia) and were distributed with their hosts worldwide. By contrast, two additional unnamed species were restricted to Australia. The present study shows that evolutionary lineages within Pisolithus are related to the biogeographical origin of the hosts. In addition, regional floras and endemic plants could act as hosts of endemic species of Pisolithus.

ITS–RFLP and ITS sequence diversity in Pisolithus from central and eastern Australian sclerophyll forests

Fifty-three isolates of Pisolithus were obtained from various locations in central and eastern Australia. These, along with two isolates from south-east Asia and one from USA, were compared using ITS–RFLP and ITS sequencing analyses. Results of the RFLP analysis initially grouped the isolates into eight RFLP types. ITS sequences were obtained for at least one representative isolate from each RFLP type and compared by a neighbour-joining analysis with Pisolithus ITS sequences available in databases. The majority of isolates clustered into four groups within two major clades, each clade comprising isolates of similar basidiospore characteristics. Most Australian isolates corresponded with recent provisional descriptions of P. albus or P. marmoratus. One isolate (LJ30) had low sequence identity (61.6-78%) to the other isolates and probably represents a separate undescribed Australian species. Significant intraspecific variation was observed in ITS–RFLP profiles for the putative P. albus isolates, suggesting that the sole use of RFLP analysis in diversity assessment may overestimate Pisolithus species richness.

Influence of salinity on biomass production by Australian Pisolithus spp. isolates

Eighteen isolates, comprising three putative Pisolithus species from eastern and central Australia, were screened for resistance to NaCl during growth in axenic culture. Biomass yield for most isolates showed a decline with increasing NaCl up to 200 mM; however, only five isolates reached an EC50 point (effective concentration inhibiting growth by 50%) below 200 mM NaCl. Most Pisolithus isolates were thus found to be resistant to NaCl at concentrations found in very saline soils. Of five isolates screened for Na2SO4 resistance, none reached an EC50 point below 100 mM. While intraspecific variation was evident in the resistance of isolates to both salts, no obvious patterns of interspecific variation were observed. The data thus indicate that isolates of the three Australian Pisolithus species are broadly resistant to salinity and may represent useful ectomycorrhizal inoculants for outplanting of compatible, salt-resistant host trees at saline sites.

Distribution and persistence of Australian Pisolithus species genets at native sclerophyll forest field sites

Basidiomes of a Pisolithus species were collected from a ca 2500 m2 Australian sclerophyll forest site during 1997 and 1999 and of a second Pisolithus species from a further ca 150 m2 site during 1999. Inter-simple sequence repeat (ISSR) PCR was conducted on DNA extracted from each basidiome using the primers 5’BDB(ACA)5, 5’DDB(CCA)5 and 5’DHB(CGA)5. Thirty-seven genotypes of Pisolithus species I were detected at the North Wilberforce site, with eight genotypes present during both 1997 and 1999. All other genotypes were observed during only one year. Mapping genotype distribution according to location of basidiome collection at the site suggested that most genotypes were present as small ( 4.0 m2) genets (including one ca 120 m2 genet) were also present. All Pisolithus species II basidiomes at the North Turramurra site were of a common genotype, suggesting the presence of a single large (ca 69 m2) below-ground mycelial genet of this taxon at the site. Both Pisolithus species thus appear to produce large long-lived soil-borne mycelia, but establishment of large genets may be restricted under some circumstances.

Distribution and persistence of Australian Pisolithus species genets at native sclerophyll forest field sites

Basidiomes of a Pisolithus species were collected from a ca 2500 m2 Australian sclerophyll forest site during 1997 and 1999 and of a second Pisolithus species from a further ca 150 m2 site during 1999. Inter-simple sequence repeat (ISSR) PCR was conducted on DNA extracted from each basidiome using the primers 5’BDB(ACA)5, 5’DDB(CCA)5 and 5’DHB(CGA)5. Thirty-seven genotypes of Pisolithus species I were detected at the North Wilberforce site, with eight genotypes present during both 1997 and 1999. All other genotypes were observed during only one year. Mapping genotype distribution according to location of basidiome collection at the site suggested that most genotypes were present as small (< 2.0 m2) below-ground mycelial genets, however several larger (> 4.0 m2) genets (including one ca 120 m2 genet) were also present. All Pisolithus species II basidiomes at the North Turramurra site were of a common genotype, suggesting the presence of a single large (ca 69 m2) below-ground mycelial genet of this taxon at the site. Both Pisolithus species thus appear to produce large long-lived soil-borne mycelia, but establishment of large genets may be restricted under some circumstances.

Characteristics of glutamine uptake by two Australian Pisolithus species

Uptake of 14 C-labelled glutamine by seven Australian Pisolithus isolates (representing two species) was investigated over the concentration range 0.5 mmol m −3 -20 mol m −3 . Total uptake did not conform to simple Michaelis-Menten kinetics over this concentration range. At concentrations above 0.5 mol m −3 much of the glutamine uptake appeared to be diffusion-like. At concentrations below 0.5 mol m −3 , subtraction of uptake in the presence of 2,4-dinitrophenol from total uptake revealed an active component that followed Michaelis-Menten kinetics. Estimated K m and V max values for active glutamine uptake by all isolates were in the ranges 4.0 - 210 mmol m −3 and 80 - 637 nmol g −1 d wt. min −1 respectively. pH optima for glutamine uptake were in the ranges 4.5-6.0, 3.7-5.0 or 3.0-4.5, depending on the isolate. While intraspecific variation was observed, there were no apparent relationships between the two Pisolithus species and either kinetic parameters for glutamine uptake or the pH optimum for the process.

Characteristics of glutamine uptake by two Australian Pisolithus species

Uptake of 14 C-labelled glutamine by seven Australian Pisolithus isolates (representing two species) was investigated over the concentration range 0.5 mmol m −3 -20 mol m −3 . Total uptake did not conform to simple Michaelis-Menten kinetics over this concentration range. At concentrations above 0.5 mol m −3 much of the glutamine uptake appeared to be diffusion-like. At concentrations below 0.5 mol m −3 , subtraction of uptake in the presence of 2,4-dinitrophenol from total uptake revealed an active component that followed Michaelis-Menten kinetics. Estimated K m and V max values for active glutamine uptake by all isolates were in the ranges 4.0 - 210 mmol m −3 and 80 - 637 nmol g −1 d wt. min −1 respectively. pH optima for glutamine uptake were in the ranges 4.5-6.0, 3.7-5.0 or 3.0-4.5, depending on the isolate. While intraspecific variation was observed, there were no apparent relationships between the two Pisolithus species and either kinetic parameters for glutamine uptake or the pH optimum for the process.

ITS sequences and mitochondrial DNA polymorphism in Pisolithus isolates

Genetic variability among isolates of the ectomycorrhizal fungus genus Pisolithus collected in Brazil, USA and France was examined, based on nucleotide sequence of ITS from rDNA genes and restriction polymorphism of the mitochondrial DNA (mtDNA). Alignment of the ITS sequences showed 97.0% homology among isolates from Brazil, 97.8% homology among isolates from USA and France and 75.7-78.5% among the isolates from Brazil and USA/France. These sequences were aligned with ITS sequences of Australian, Brazilian and Kenyan Pisolithus isolates described in the literature. A consensus tree generated by sequence data of all Pisolithus isolates showed the presence of a number of species among the isolates. The mtDNA analysis was carried out by total DNA restriction analysis with different enzymes ( Dra I, EcoR I, EcoR V, Hha I- Xba I, Hind III and Rsa I) and probing with purified mtDNA labelled with fluorescein d-UTP. The size of the mitochondrial genome of isolates Pt 90A, Pt 185R and PF cleaved with EcoR I was estimated to be, approx. 40.5, 47.2 and 47.6 kb, respectively. Cluster analysis based on the mtDNA restriction fragments grouped the isolates into two distinct groups, which coincide with their host specificity and geographical origins. PCR-RFLP analysis of mitochondrial rDNA (mt rDNA) from 12 Pisolithus isolates did not show any polymorphism. These data corroborate previous results obtained in our laboratory with RAPD-PCR and rDNA PCR-RFLP markers and support recent taxonomic studies where a number of Pisolithus species were described.

Intra- and interspecific variation in patterns of organic and inorganic nitrogen utilization by three Australian Pisolithus species

The ability of three Australian Pisolithus species, discriminated on the basis of ITS sequence data, to utilize a range of inorganic and organic nitrogen sources was assessed in liquid axenic culture. Both intra-, and putative interspecific, variation in nitrogen source utilization was observed. Most isolates demonstrated a preference for NH4+ over NO3−, although some showed no significant preference for either inorganic source. All isolates utilized a range of amino acids. Species I isolates demonstrated a preference for acidic and/or neutral amino acids over basic acids, while species II and III isolates generally utilized amino acids poorly relative to species I. Although most isolates utilized BSA poorly, two species I isolates that had been maintained in axenic culture for > 10 y grew well on this substrate, suggesting possible changes in nitrogen utilization with extended storage in axenic culture.