Armillaria altimontana in North America: Biology and Ecology.

Armillaria altimontana is a fungus (Basidiomycota, Agaricomycetes, Agaricales, and Physalacriaceae) that is generally considered as a weak/opportunistic pathogen or saprophyte on many tree hosts. It widely occurs across the northwestern USA to southern British Columbia, Canada, but relatively little is known about its ecological role in the diverse forest ecosystems where it occurs. This review summarizes the biology and ecology of A. altimontana, including its identification, life cycle, distribution, host associations, and bioclimatic models under climate change.

First Report of the Armillaria Root Disease Pathogen, Armillaria solidipes, on Black Oak (Quercus velutina) in North Carolina, USA.

Armillaria root disease is among the largest causes of mortality and lost productivity of widely ranging horticultural, urban, and forest trees/shrubs in diverse boreal, temperate, and tropical regions around the world (Kim et al. 2022). Damage from Armillaria root disease will likely increase in response to changing climate and extreme weather because environmental stress can predispose host trees to Armillaria root disease (Murray and Leslie 2021; Kim et al. 2021). On August 14, 2021, a mature black oak (Quercus velutina) ca. 29 m in height and 64 cm DBH experienced a complete structural failure originating at the root plate, falling onto a major highway within the Pisgah National Forest near Brevard, North Carolina (35°16'50.876"N, 82°42'51.785"W, elevation 680 m) during sunny, calm conditions. No above-ground indications of predisposing disturbance, injury, or insect/disease activity were observed. Advanced wood decay, however, was present in many structural roots of the exposed root plate; abundant rhizomorphs attached to the surfaces of most roots were noted (Supplemental Figure 1). One large primary root (ca. 23 cm in diameter) exhibited clear signs of Armillaria root disease at the point of failure as evidenced by an extensive decay column, abundant black rhizomorphs on the root surface, and white mycelial fans (a sign of pathogenicity) within the root itself (Supplemental Figure 1). Rhizomorph samples were established in culture, two Armillaria isolates (PNF#001R-1 and PNF#001R-2) were examined, and pairing tests showed that both isolates belonged to the same genet (PNF#001R-1 = PNF#001R-2). Based on translation elongation factor 1α (tef1) sequences, both isolates (one genet) were identified as A. solidipes (GenBank accession no. OP823701), showing 98% similarity with A. solidipes tef1 sequences (e.g., MH879015) in GenBank. Also, nine replications of somatic incompatibility tests of the isolate with five North American Armillaria spp. [A. solidipes, A. mellea, A. gallica, A. mexicana, and Desarmillaria caespitosa (= North American A. tabescens)] showed 67% compatibility with A. solidipes, compared with 0-22% for A. mellea, D. caespitosa, A. gallica, and A. mexicana. All test species but A. mexicana are reported to occur in the eastern and/or southern USA, while A. mexicana occurs in a similar environment in Mexico (Elías-Román et al. 2018). To our knowledge, this is the first confirmed report of A. solidipes in North Carolina, USA, where it was causing disease on black oak, and this is the most southerly confirmed case of A. solidipes in eastern North America. Although Armillaria inoculation tests are impractical, black oak was previously reported as a host of A. solidipes in Massachusetts, USA (Brazee and Wick 2009). Armillaria solidipes is considered as the most important Armillaria root disease pathogen of conifer forests in western USA (Lockman and Kearns 2016), but it has been suggested that A. solidipes can thrive in northern hardwood forests that reside near conifer forests (Brazee and Wick 2009). Climate change is predicted to increase damage caused by A. solidipes on conifers in the western USA (Kim et al. 2021); however, it is undetermined if the occurrence of A. solidipes-caused disease in North Carolina is related to climate change or how climate change could influence this disease across the region. More surveys are needed to assess the impact of Armillaria root disease on health of mixed forests in the eastern USA.

Genomic Comparisons of Two Armillaria Species with Different Ecological Behaviors and Their Associated Soil Microbial Communities.

Armillaria species show considerable variation in ecological roles and virulence, from mycorrhizae and saprophytes to important root pathogens of trees and horticultural crops. We studied two Armillaria species that can be found in coniferous forests of northwestern USA and southwestern Canada. Armillaria altimontana not only is considered as a weak, opportunistic pathogen of coniferous trees, but it also appears to exhibit in situ biological control against A. solidipes, formerly North American A. ostoyae, which is considered a virulent pathogen of coniferous trees. Here, we describe their genome assemblies and present a functional annotation of the predicted genes and proteins for the two Armillaria species that exhibit contrasting ecological roles. In addition, the soil microbial communities were examined in association with the two Armillaria species within a 45-year-old plantation of western white pine (Pinus monticola) in northern Idaho, USA, where A. altimontana was associated with improved tree growth and survival, while A. solidipes was associated with reduced growth and survival. The results from this study reveal a high similarity between the genomes of the beneficial/non-pathogenic A. altimontana and pathogenic A. solidipes; however, many relatively small differences in gene content were identified that could contribute to differences in ecological lifestyles and interactions with woody hosts and soil microbial communities.

Translocation of Fungicides and Their Efficacy in Controlling Phellinus noxius, the Cause of Brown Root Rot Disease.

Brown root rot disease (BRRD), caused by Phellinus noxius, is an important tree disease in tropical and subtropical areas. To improve chemical control of BRRD and deter emergence of fungicide resistance in P. noxius, this study investigated control efficacies and systemic activities of fungicides with different modes of action. Fourteen fungicides with 11 different modes of action were tested for inhibitory effects in vitro on 39 P. noxius isolates from Taiwan, Hong Kong, Malaysia, Australia, and Pacific Islands. Cyproconazole, epoxiconazole, and tebuconazole (Fungicide Resistance Action Committee [FRAC] 3, target-site G1) inhibited colony growth of P. noxius by 99.9 to 100% at 10 ppm and 97.7 to 99.8% at 1 ppm. The other effective fungicide was cyprodinil + fludioxonil (FRAC 9 + 12, target-site D1 + E2), which showed growth inhibition of 96.9% at 10 ppm and 88.6% at 1 ppm. Acropetal translocation of six selected fungicides was evaluated in bishop wood (Bischofia javanica) seedlings by immersion of the root tips in each fungicide at 100 ppm, followed by liquid or gas chromatography tandem mass spectrometry analyses of consecutive segments of root, stem, and leaf tissues at 7 and 21 days posttreatment. Bidirectional translocation of the fungicides was also evaluated by stem injection of fungicide stock solutions. Cyproconazole and tebuconazole were the most readily absorbed by roots and efficiently transported acropetally. Greenhouse experiments suggested that cyproconazole, tebuconazole, and epoxiconazole have a slightly higher potential for controlling BRRD than mepronil, prochloraz, and cyprodinil + fludioxonil. Because all tested fungicides lacked basipetal translocation, soil drenching should be considered instead of trunk injection for their use in BRRD control.

Phylogenetic analyses allow species-level recognition of Leptographium wageneri varieties that cause black stain root disease of conifers in western North America.

Leptographium wageneri is a native fungal pathogen in western North America that causes black stain root disease (BSRD) of conifers. Three host-specialized varieties of this pathogen were previously described: L. wageneri var. wageneri on pinyon pines (Pinus monophylla and P. edulis); L. wageneri var. ponderosum, primarily on hard pines (e.g., P. ponderosa, P. jeffreyi); and L. wageneri var. pseudotsugae on Douglas-fir (Pseudotsuga menziesii). Morphological, physiological, and ecological differences among the three pathogen varieties have been previously determined; however, DNA-based characterization and analyses are needed to determine the genetic relationships among these varieties. The objective of this study was to use DNA sequences of 10 gene regions to assess phylogenetic relationships among L. wageneri isolates collected from different hosts. The multigene phylogenetic analyses, based on maximum likelihood and Bayesian inference, strongly supported species-level separation of the three L. wageneri varieties. These results, in conjunction with previously established phenotypic differences, support the elevation of L. wageneri var. ponderosum and L. wageneri var. pseudotsugae to the species level as L. ponderosum comb. nov. and L. pseudotsugae comb. nov., respectively, while maintaining L. wageneri var. wageneri as Leptographium wageneri. Characterization of the three Leptographium species, each with distinct host ranges, provides a baseline to further understand the ecological interactions and evolutionary relationships of these forest pathogens, which informs management of black stain root disease.

First Report of Armillaria root disease of Celtis laevigata caused by A. gallica in South Carolina, USA.

Celtis laevigata (sugarberry, southern hackberry) is an important, shade-tolerant, deciduous hardwood tree species that occurs naturally in flood plains, along streams and rivers, and in urban landscapes of the southeastern USA (Kennedy 1990). In recent years, dieback and mortality of C. laevigata have been commonly observed in some areas of South Carolina (SC) and Georgia (GA) (Poole et al. 2021). In April/May of 2018, the crown conditions and root systems were examined for three C. laevigata trees in North Augusta, SC. The crown of each tree was visually assessed using the method of Poole et al. (2021). Root samples were obtained by excavating two main roots ca. 2 meters away from the stem of each tree. Tree SB474 (N33 29.472, W81 59.082, elev. 55.8 m) exhibited > 66% crown loss and decaying roots with white mycelial fans and dark rhizomorphs characteristic of Armillaria. Tree SB913 (N33 29.830, W81 59.349, elev. 58.8 m) exhibited ca. 34-66% crown loss, while tree SB914 (N33 29.837, W81 59.338, elev. 57 m) appeared healthy with no apparent crown loss. Roots of trees SB913 and SB914 appeared healthy, although rhizomorphs were attached to the root surfaces. Roots and/or attached rhizomorphs were surface disinfested and plated n a basidiomycete-selective medium (Hendrix and Kuhlman 1962). Three Armillaria isolates, one from each corresponding tree, were paired with each other, and two genets were identified (SB474 and SB913 = SB914). The two genets (SB474 and SB913) were used in somatic pairing tests against three known tester isolates for each of the following species: A. solidipes, A. mellea, A. gallica, A. mexicana, and Desarmillaria caespitosa (=A. tabescens). Pairing of isolates SB474 and SB913 showed the highest compatibility with A. gallica (isolates ST22, ST23, and M70) with 100% and 89%, respectively. These isolates were definitively confirmed as A. gallica by translation elongation factor 1α gene sequences (tef1; Klopfenstein et al. 2017) (GenBank accession nos. OM993577 and OM993578 for SB474 and SB913, respectively). GenBank nucleotide BLAST showed tef1 similarity for both SB474 and SB913 isolates was highest for A. gallica (≥98.7%; GenBank accession nos. MT761696, MT761697, and KF156772). This is the first report of A. gallica associated with Armillaria root disease of C. laevigata. Rhizomorphs on the surface of apparently healthy tree roots and root colonization in severely declining trees are a common sign of A. gallica (Baumgartner and Rizzo 2001). Pathogen colonization of root surfaces may provide an opportunity for infection of highly damaged trees, resulting in root disease (Gregory 1985). Primary agents of C. laevigata dieback and mortality in SC and GA remain undefined, but continued study is needed to confirm the role of A. gallica in C. laevigata dieback and mortality. Although pathogenicity tests are impractical for Armillaria, these A. gallica occurrences in SC further adds to our knowledge of this pathogen's distribution in the southeastern USA, where it has also been confirmed in Tennessee in hardwood forests (Bruhn et al. 1997), SC on Hemerocallis sp. (Schnabel et al. 2005), and GA on a Rhododendron/span> sp. and Quercus rubra (Hanna et al. 2020). The distribution and host range of A. gallica is likely more widespread in the southeastern USA than existing records indicate. Documenting Armillaria distribution, including A. gallica, is essential for predicting climate-change impacts on Armillaria root diseases (Kim et al. 2022). Baumgartner, K., and Rizzo, D. M. 2001. Plant Dis. 85:947-951. Bruhn, J. N., et al. 1997. In 11th Central Hardwood Forest Conference, USDA, FS, NC-GTR-188, 49-57. Gregory, S. C. 1985. Plant Path. 34:41-48. Hanna, J. W., et al. 2020. Plant Dis. 105: 1226. Hendrix Jr, F. F., and Kuhlman, E. G. 1962. PI. Dis. Rep. 46:674-676. Kennedy, Jr., H. E. 1990. Silvics of North America: 2. Hardwoods. USDA-FS. Agriculture Handbook 654. Kim, M.-S., et al. 2022. Front. For. Glob. Change 4:740994. Klopfenstein, N. B., et al. 2017. Mycologia 109:75-91. Poole, E. M., et al. 2021. J. For. 119:266-274. Schnabel, G., et al. 2005. Plant Dis. 89:683.

Long evolutionary history of an emerging fungal pathogen of diverse tree species in eastern Asia, Australia and the Pacific Islands.

Emerging plant pathogens have been increasing exponentially over the last century. To address this issue, it is critical to determine whether these pathogens are native to ecosystems or have been recently introduced. Understanding the ecological and evolutionary processes fostering emergence can help to manage their spread and predict epidemics/epiphytotics. Using restriction site-associated DNA sequencing data, we studied genetic relationships, pathways of spread and the evolutionary history of Phellinus noxius, an emerging root-rotting fungus of unknown origin, in eastern Asia, Australia and the Pacific Islands. We analysed patterns of genetic variation using Bayesian inference, maximum-likelihood phylogeny, population splits and mixtures measuring correlations in allele frequencies and genetic drift, and finally applied coalescent-based theory using Approximate Bayesian computation (ABC) with supervised machine learning. Population structure analyses revealed five genetic groups with signatures of complex recent and ancient migration histories. The most probable scenario of ancient pathogen spread is movement from an unsampled population to Malaysia and the Pacific Islands, with subsequent spread to Taiwan and Australia. Furthermore, ABC analyses indicate P. noxius spread occurred thousands of generations ago, contradicting previous assumptions that this pathogen was recently introduced to multiple geographical regions. Our results suggest that recent emergence of P. noxius in eastern Asia, Australia and the Pacific Islands has probably been driven by anthropogenic and natural disturbances, such as deforestation, land-use change, severe weather events and/or introduction of exotic plants. This study provides a novel example of applying genome-wide allele frequency data to unravel the dynamics of pathogen emergence under changing ecosystem conditions.

Desarmillaria caespitosa, a North American vicariant of D. tabescens.

Desarmillaria caespitosa, a North American vicariant species of European D. tabescens, is redescribed in detail based on recent collections from the USA and Mexico. This species is characterized by morphological features and multilocus phylogenetic analyses using portions of nuc rDNA 28S (28S), translation elongation factor 1-alpha (tef1), the second largest subunit of RNA polymerase II (rpb2), actin (act), and glyceraldehyde-3-phosphate dehydrogenase (gpd). A neotype of D. caespitosa is designated here. Morphological and genetic differences between D. caespitosa and D. tabescens were identified. Morphologically, D. caespitosa differs from D. tabescens by having wider basidiospores, narrower cheilocystidia, which are often irregular or mixed (regular, irregular, or coralloid), and narrower caulocystidia. Phylogenetic analyses of five independent gene regions show that D. caespitosa and D. tabescens are separated by nodes with strong support. The new combination, D. caespitosa, is proposed.

First report of Armillaria cepistipes causing root disease on Populus trichocarpa (black cottonwood) in Oregon, USA.

Populus trichocarpa Torr. and Gray (black cottonwood) is an economically and ecologically important tree species native to western North America. It serves as a model tree species in biology and genetics due to its relatively small genome size, rapid growth, and early reproductive maturity (Jansson and Douglas 2007). Black cottonwood is susceptible to root rot caused by at least one species of Armillaria (Raabe 1962), a globally distributed genus that exhibits diverse ecological behaviors (Klopfenstein et al. 2017) and infects numerous woody plant species (Raabe 1962). However, several Armillaria spp. have been isolated from Populus spp. in North America (Mallet 1990), and the most recent report of Armillaria on P. trichocarpa used the now ambiguated name A. mellea (Vahl.) Quel. (see Raabe 1962). In April 2016, mycelial fans and rhizomorphs of an unknown Armillaria species (isolate WV-ARR-3) were collected from P. trichocarpa in a riparian hardwood stand ca. 5.5 km east of Springfield, Oregon, USA (44°3'21.133"N, 122°49'39.935"W). The host was dominant in the canopy, large in diameter (ca. 90-cm dbh) relative to neighboring trees, and exhibited minimal crown dieback (ca. < 5%). A mycelial fan was observed destroying living cambium beneath the inner bark, indicating pathogenicity. The isolate was cultured on malt extract medium (3% malt extract, 3% dextrose, 1% peptone, and 1.5 % agar) and identified as A.cepistipes on the basis of somatic pairing tests and translation elongation factor 1α (tef1) sequences (GenBank Accession No. MK172784). DNA extraction, PCR, and tef1 sequencing followed protocols of Elías-Román et al. (2018). From nine replications of somatic incompatibility tests (18 tester isolates representing six North American Armillaria spp.), the isolate showed high intraspecific compatibility (colorless antagonism) with three A. cepistipes tester isolates (78%), but low compatibility with the other Armillaria spp. (0 - 33%) that occur in the region. Isolate WV-ARR-3 yielded tef1 sequences with a 99% identity to A. cepistipes (GenBank Accession Nos. JF313115 and JF313121). A second isolate (WV-ARR-1; GenBank Accession No. MK172783) with a nearly identical sequence was collected from a maturing P. trichocarpa in a riparian stand ca. 8 km northeast of Monroe, Oregon (44°21'47.57"N, 123°13'14.415"W) along the Willamette River, downstream from the McKenzie river tributary where WV-ARR-3 was collected. Armillaria cepistipes has been reported on Alnus rubra (red alder) in Washington, USA (Banik et al. 1996) and on broad-leaved trees in British Columbia, Canada (Allen et al. 1996). It is generally considered to be a weak pathogen on broad-leaved trees in the Pacific Northwest, but it is also associated with pathogenicity on both coniferous and deciduous trees in Europe (e.g., Lygis et al. 2005). However, a recent phylogenetic study suggested that North American A. cepistipes is phylogenetically distinct from Eurasian A. cepistipes (Klopfenstein et al. 2017), butadditional studies are needed to determine the formal taxonomic status of North American A. cepistipes. To our knowledge, A. cepistipes has not been previously confirmed on P. trichocarpa in the U.S.A. or formally reported as a pathogen of any Populus species in North America. Continued studies are needed to determine the distribution, host range, and ecological role of A. cepistipes in riparian forests of the Pacific Northwest, while monitoring its populations under changing climates.

First Report of Armillaria Root Disease Pathogen, Armillaria gallica, on Rhododendron and Quercus rubra in Georgia, USA.

Armillaria root and butt diseases, which are a global issue, can be influenced by changing environmental conditions. Armillaria gallica is a well-known pathogen of diverse trees worldwide (Brazee and Wick 2009). Besides A. gallica causing root rot of Hemerocallis sp. and Cornus sp. in South Carolina (Schnabel et al. 2005), little is reported on the distribution and host range of A. gallica in the southeastern USA. In July 2017, three Armillaria isolates were obtained from two naturally occurring hosts in Georgia, USA and cultured on malt extract medium (3% malt extract, 3% dextrose, 1% peptone, and 1.5% agar). One isolate (GA3) was obtained in Unicoi State Park near Helen, Georgia (Lat. 34.712275, Long. -83.727765, elev. 498 m) from the basal portion of Rhododendron sp. with extensive root/butt decay, but no crown symptoms were evident (Supplementary Figure 1). GA4 and GA5 (Lat. 33.902433, Long. -83.382453, elev. 215 m) were isolated from wind-felled Quercus rubra (red oak) with root disease at the State Botanical Gardens in Athens, Georgia. GA4 was associated with a large root ball (ca. 4-m diameter) (Supplementary Figure 2), and GA5 was obtained from a mature tree with infected roots, with characteristic spongy rot of Armillaria root disease. Crown symptoms could not be evaluated because the crowns had been removed before the collections. Several other oaks with Armillaria root disease were noted throughout the State Botanical Gardens. Pairing tests reduced these three isolates (whiteish mycelia with a dark, brownish crust and rhizomorphs), to two genets with GA4 = GA5. Both genets (GA3 and GA4) were identified as A. gallica using translation elongation factor 1α (tef1) sequences (Genbank Nos. MT761697 and MT761698, respectively) that showed ≥ 97% identity (≥ 98% coverage) with A. gallica sequences (KF156772, KF156775). Also, nine replications of somatic pairing tests showed 33 - 67% compatibility with A. gallica (occurs in southeastern USA), compared with 0 - 22% for A. mexicana, A. mellea (occurs in southeastern USA), A. solidipes, and Desarmillaria tabescens (occurs in southeastern USA). To our knowledge, this note represents the first report of A. gallica on Rhododendron and Q. rubra in Georgia, USA, which has experienced severe drought in recent decades (e.g., Park Williams et al. 2017) that could predispose trees to Armillaria infection (e.g., Wargo 1996). Quercus rubra was previously reported as a host of A. gallica in Arkansas (Kelley et al. 2009) and Massachusetts (Brazee and Wick 2009), USA. In Missouri, USA, A. gallica has been reported as a weak pathogen with potential biological control against A. mellea (Bruhn et al. 2000). Other reports from several regions on various hosts suggest pathogenicity of A. gallica is associated with changing climate (Nelson et al. 2013, Kim et al. 2017, Kubiak et al. 2017). Wide genetic variation and/or cryptic speciation within A. gallica may account for differences in ecological behavior (Klopfenstein et al. 2017), but this is difficult to evaluate because Armillaria pathogenicity tests cannot be used on most forest tree seedlings. This study suggests that A. gallica is more widespread than previously known and its adverse impacts on woody plants may intensify over time, depending on the environmental conditions. Further studies are needed to determine environmental influences on A. gallica, the full distribution of A. gallica, and its effects in forests of the southeastern USA.

Whole genome analysis of the koa wilt pathogen (Fusarium oxysporum f. sp. koae) and the development of molecular tools for early detection and monitoring.

BACKGROUND: Development and application of DNA-based methods to distinguish highly virulent isolates of Fusarium oxysporum f. sp. koae [Fo koae; cause of koa wilt disease on Acacia koa (koa)] will help disease management through early detection, enhanced monitoring, and improved disease resistance-breeding programs. RESULTS: This study presents whole genome analyses of one highly virulent Fo koae isolate and one non-pathogenic F. oxysporum (Fo) isolate. These analyses allowed for the identification of putative lineage-specific DNA and predicted genes necessary for disease development on koa. Using putative chromosomes and predicted gene comparisons, Fo koae-exclusive, virulence genes were identified. The putative lineage-specific DNA included identified genes encoding products secreted in xylem (e. g., SIX1 and SIX6) that may be necessary for disease development on koa. Unique genes from Fo koae were used to develop pathogen-specific PCR primers. These diagnostic primers allowed target amplification in the characterized highly virulent Fo koae isolates but did not allow product amplification in low-virulence or non-pathogenic isolates of Fo. Thus, primers developed in this study will be useful for early detection and monitoring of highly virulent strains of Fo koae. Isolate verification is also important for disease resistance-breeding programs that require a diverse set of highly virulent Fo koae isolates for their disease-screening assays to develop disease-resistant koa. CONCLUSIONS: These results provide the framework for understanding the pathogen genes necessary for koa wilt disease and the genetic variation of Fo koae populations across the Hawaiian Islands.

First Report of the Armillaria Root-Disease Pathogen, Armillaria gallica, Associated with Several Woody Hosts in Three States of Central Mexico (Guanajuato, Jalisco, and Michoacan).

In July-August 2019, seven Armillaria isolates (derived from rhizomorphs and mycelial fans of infected roots) were collected in association with woody hosts in the central Mexico: states of Guanajuato (MEX204), Jalisco (MEX206, MEX208, MEX209), and Michoacan (MEX211, MEX214, MEX216). All seven isolates were identified as Armillaria gallica based on translation elongation factor 1α (tef1) gene sequences (GenBank accession Nos.: MN839636 - MN839642 for MEX204, MEX206, MEX208, MEX209, MEX211, MEX214, and MEX216) and somatic pairing tests against known tester isolates. GenBank nucleotide BLAST results showed tef1 similarity for all isolates was highest for with A. gallica (≥ 97%; GenBank Accession Nos. KF156775 and KF156772). In replicated pairings against three tester isolates each for A. gallica, A. mellea, and A. mexicana, all isolates showed the highest compatibility with A. gallica (67-100%), with lower compatibility against A. mellea and A. mexicana, with 3-11% and 2-11%, respectively. Variations in compatibility among different tester isolates could reflect cryptic speciation within A. gallica (Klopfenstein et al., 2017). In Tarimoro, Guanajuato, MEX204 was isolated from infected Quercus jonesii (20°13'46.2"N 100°42'51.1"W, elevation 2286 m) that displayed root disease symptoms/signs (wilting/defoliation and mycelial fans within the roots). In a forested area of Mazamitla, Jalisco, MEX206 was isolated from infected Quercus laevis (19°54´52"N 103°00´07"W, elevation 2564 m) with root disease symptoms/signs (e.g., wilting, foliar chlorosis, and mycelial fans within the root crown); MEX208 was isolated from infected Pinus pseudostrobus (19°54´53"N 102°59´54"W, elevation 2554 m) with basal resinosis and mycelial fans; and MEX209 was collected from a symptomless P. devoniana (19°54'13.1"N 103°00'14.1"W, elevation 2566 m). In Zinapecuaro, Michoacan, MEX211 (19°53'28.8"N 100°39'44.0"W, elevation 2587 m) was isolated from infected Malus domestica with root disease that resulted in mortality; in Hidalgo, Michoacan, MEX214 (19°46'49"N 100°39'25.2"W, elevation 2961 m) and MEX216 (19°46'58"N 100°39'24"W, elevation 2958 m) were isolated from infected P. devoniana and P. teocote, respectively, which both displayed root disease symptoms/signs (basal resinosis and mycelial fans). Previously, A. gallica was reported in the State of Mexico, Veracruz, Oaxaca, Mexico (Elías-Román et al. 2013; Klopfenstein et al. 2014), but this represents the first report of A. gallica in Guanajuato, Jalisco, and Michoacan, Mexico. In contrast to other regions of North America (e.g., Bruhns et al. 2000), A. gallica was demonstrated to be a virulent pathogen on peach (Prunus persica) in central Mexico (Elías-Román et al. 2013). Unfortunately, tree seedlings cannot be used for Armillaria pathogenicity tests in a greenhouse or nursery; however, all root-diseased trees in this report showed Armillaria mycelial fans under the bark of a living tree, which are reliable indicators of pathogenicity, and no other root diseases were found. This report demonstrates that A. gallica is distributed across central Mexico, where it is associated with disease on Quercus, Pinus, and Malus. Such information is critical to increase our understanding of Armillaria root disease across diverse geographic regions and climates.

Genome comparison and transcriptome analysis of the invasive brown root rot pathogen, Phellinus noxius, from different geographic regions reveals potential enzymes associated with degradation of different wood substrates.

Phellinus noxius is a root-decay pathogen with a pan-tropical/subtropical distribution that attacks a wide range of tree hosts. For this study, genomic sequencing was conducted on P. noxius isolate P919-02W.7 from Federated States of Micronesia (Pohnpei), and its gene expression profile was analyzed using different host wood (Acer, Pinus, Prunus, and Salix) substrates. The assembled genome was 33.92 Mbp with 2954 contigs and 9389 predicted genes. Only small differences were observed in size and gene content in comparison with two other P. noxius genome assemblies (isolates OVT-YTM/97 from Hong Kong, China and FFPRI411160 from Japan, respectively). Genome analysis of P. noxius isolate P919-02W.7 revealed 488 genes encoding proteins related to carbohydrate and lignin metabolism, many of these enzymes are associated with degradation of plant cell wall components. Most of the transcripts expressed by P. noxius isolate P919-02W.7 were similar regardless of wood substrates. This study highlights the vast suite of decomposing enzymes produced by P. noxius, which suggests potential for degrading diverse wood substrates, even from temperate host trees. This information contributes to our understanding of pathogen ecology, mechanisms of wood decomposition, and pathogenic/saprophytic lifestyle.

Genomic comparisons of the laurel wilt pathogen, Raffaelea lauricola, and related tree pathogens highlight an arsenal of pathogenicity related genes.

Raffaelea lauricola is an invasive fungal pathogen and symbiont of the redbay ambrosia beetle (Xyleborus glabratus) that has caused widespread mortality to redbay (Persea borbonia) and other Lauraceae species in the southeastern USA. We compare two genomes of R. lauricola (C2646 and RL570) to seven other related Ophiostomatales species including R. aguacate (nonpathogenic close relative of R. lauricola), R. quercus-mongolicae (associated with mortality of oaks in Korea), R. quercivora (associated with mortality of oaks in Japan), Grosmannia clavigera (cause of blue stain in conifers), Ophiostoma novo-ulmi (extremely virulent causal agent of Dutch elm disease), O. ulmi (moderately virulent pathogen that cause of Dutch elm disease), and O. piceae (blue-stain saprophyte of conifer logs and lumber). Structural and functional annotations were performed to determine genes that are potentially associated with disease development. Raffaelea lauricola and R. aguacate had the largest genomes, along with the largest number of protein-coding genes, genes encoding secreted proteins, small-secreted proteins, ABC transporters, cytochrome P450 enzymes, CAZYmes, and proteases. Our results indicate that this large genome size was not related to pathogenicity but was likely lineage specific, as the other pathogens in Raffaelea (R. quercus-mongolicae and R. quercivora) had similar genome characteristics to the Ophiostoma species. A diverse repertoire of wood-decaying enzymes were identified in each of the genomes, likely used for toxin neutralization rather than wood degradation. Lastly, a larger number of species-specific, secondary metabolite, synthesis clusters were identified in R. lauricola suggesting that it is well equipped as a pathogen, which could explain its success as a pathogen of a wide range of lauraceous hosts.

Armillaria mexicana, a newly described species from Mexico.

Armillaria mexicana (Agaricales, Physalacriaceae) is described as a new species based on morphology, DNA sequence data, and phylogenetic analyses. It clearly differs from previously reported Armillaria species in North, Central, and South America. It is characterized by the absence of fibulae in the basidioma, abundant cheilocystidia, and ellipsoidal, hyaline basidiospores that are apparently smooth under light microscope, but slightly to moderately rugulose under scanning electron microscope. It is differentiated from other Armillaria species by macromorphological characters, including annulus structure, pileus and stipe coloration, and other structures. DNA sequence data (nuc rDNA internal transcribed spacers [ITS1-5.8S-ITS2 = ITS], 28S D-domain, 3' end of 28S intergenic spacer 1, and translation elongation factor 1-α [TEF1]) show that A. mexicana sequences are quite distinct from sequences of analogous Armillaria species in GenBank. In addition, sequences of ITS of the A. mexicana ex-type culture reveal an ITS1 of 1299 bp and an ITS2 of 582 bp, the longest ITS regions reported thus far in fungi. Phylogenetic analysis based on TEF1 sequences place A. mexicana in a well-separated, monophyletic clade basal to the polyphyletic A. mellea complex.

Draft Genome Sequence of the Fungus Associated with Oak Wilt Mortality in South Korea, Raffaelea quercus-mongolicae KACC44405.

The fungus Raffaelea quercus-mongolicae is the causal agent of Korean oak wilt, a disease associated with mass mortality of oak trees (e.g., Quercus spp.). The fungus is vectored and dispersed by the ambrosia beetle, Platypus koryoensis Here, we present the 27.0-Mb draft genome sequence of R. quercus-mongolicae strain KACC44405.

Insights into the phylogeny of Northern Hemisphere Armillaria: Neighbor-net and Bayesian analyses of translation elongation factor 1-α gene sequences.

Armillaria possesses several intriguing characteristics that have inspired wide interest in understanding phylogenetic relationships within and among species of this genus. Nuclear ribosomal DNA sequence-based analyses of Armillaria provide only limited information for phylogenetic studies among widely divergent taxa. More recent studies have shown that translation elongation factor 1-α (tef1) sequences are highly informative for phylogenetic analysis of Armillaria species within diverse global regions. This study used Neighbor-net and coalescence-based Bayesian analyses to examine phylogenetic relationships of newly determined and existing tef1 sequences derived from diverse Armillaria species from across the Northern Hemisphere, with Southern Hemisphere Armillaria species included for reference. Based on the Bayesian analysis of tef1 sequences, Armillaria species from the Northern Hemisphere are generally contained within the following four superclades, which are named according to the specific epithet of the most frequently cited species within the superclade: (i) Socialis/Tabescens (exannulate) superclade including Eurasian A. ectypa, North American A. socialis (A. tabescens), and Eurasian A. socialis (A. tabescens) clades; (ii) Mellea superclade including undescribed annulate North American Armillaria sp. (Mexico) and four separate clades of A. mellea (Europe and Iran, eastern Asia, and two groups from North America); (iii) Gallica superclade including Armillaria Nag E (Japan), multiple clades of A. gallica (Asia and Europe), A. calvescens (eastern North America), A. cepistipes (North America), A. altimontana (western USA), A. nabsnona (North America and Japan), and at least two A. gallica clades (North America); and (iv) Solidipes/Ostoyae superclade including two A. solidipes/ostoyae clades (North America), A. gemina (eastern USA), A. solidipes/ostoyae (Eurasia), A. cepistipes (Europe and Japan), A. sinapina (North America and Japan), and A. borealis (Eurasia) clade 2. Of note is that A. borealis (Eurasia) clade 1 appears basal to the Solidipes/Ostoyae and Gallica superclades. The Neighbor-net analysis showed similar phylogenetic relationships. This study further demonstrates the utility of tef1 for global phylogenetic studies of Armillaria species and provides critical insights into multiple taxonomic issues that warrant further study.

Rust disease of eucalypts, caused by Puccinia psidii, did not originate via host jump from guava in Brazil.

The rust fungus, Puccinia psidii, is a devastating pathogen of introduced eucalypts (Eucalyptus spp.) in Brazil where it was first observed in 1912. This pathogen is hypothesized to be endemic to South and Central America and to have first infected eucalypts via a host jump from native guava (Psidium guajava). Ten microsatellite markers were used to genotype 148 P. psidii samples from eucalypts and guava plus five additional myrtaceous hosts across a wide geographic range of south-eastern Brazil and Uruguay. Principal coordinates analysis, a Bayesian clustering analysis and a minimum-spanning network revealed two major genetic clusters among the sampled isolates, one associated with guava and another associated with eucalypts and three additional hosts. Multilocus genotypes infecting guava differed by multiple mutational steps at eight loci compared with those infecting eucalypts. Approximate Bayesian computation revealed that evolutionary scenarios involving a coalescence event between guava- and eucalypt-associated pathogen populations within the past 1000 years are highly unlikely. None of the analyses supported the hypothesis that eucalypt-infecting P. psidii in Brazil originated via host jump from guava following the introduction of eucalypts to Brazil approximately 185 years ago. The existence of host-associated biotypes of P. psidii in Brazil indicates that this diversity must be considered when assessing the invasive threat posed by this pathogen to myrtaceous hosts worldwide.

Molecular Characterization of Fusarium oxysporum and Fusarium commune Isolates from a Conifer Nursery.

ABSTRACT Fusarium species can cause severe root disease and damping-off in conifer nurseries. Fusarium inoculum is commonly found in most container and bareroot nurseries on healthy and diseased seedlings, in nursery soils, and on conifer seeds. Isolates of Fusarium spp. can differ in virulence; however, virulence and colony morphology are not correlated. Forty-one isolates of Fusarium spp., morphologically indistinguishable from F. oxysporum, were collected from nursery samples (soils, healthy seedlings, and diseased seedlings). These isolates were characterized by amplified fragment length polymorphism (AFLP) and DNA sequencing of nuclear rDNA (internal transcribed spacer including 5.8S rDNA), mitochon-drial rDNA (small subunit [mtSSU]), and nuclear translation elongation factor 1-alpha. Each isolate had a unique AFLP phenotype. Out of 121 loci, 111 (92%) were polymorphic; 30 alleles were unique to only highly virulent isolates and 33 alleles were unique to only isolates nonpathogenic on conifers. Maximum parsimony and Bayesian analyses of DNA sequences from all three regions and the combined data set showed that all highly virulent isolates clearly separated into a common clade that contained F. commune, which was recently distinguished from its sister taxon, F. oxysporum. Interestingly, all but one of the nonpathogenic isolates grouped into a common clade and were genetically similar to F. oxysporum. The AFLP cladograms had similar topologies when compared with the DNA-based phylograms. Although all tested isolates were morphologically indistinguishable from F. oxysporum based on currently available monographs, some morphological traits can be plastic and unreliable for identification of Fusarium spp. We consider the highly virulent isolates to be F. commune based on strong genetic evidence. To our knowledge, this is the first reported evidence that shows F. commune is a cause of Fusarium disease (root rot and dampingoff) on Douglas-fir seedlings. Furthermore, several AFLP genetic markers and mtSSU sequences offer potential for development of molecular markers that could be used to detect and distinguish isolates of F. oxysporum nonpathogenic to conifers and highly virulent isolates of F. commune in forest nurseries.