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Highlights:
Armillaria root disease-caused mortality of avocado trees was associated with Armillaria mexicana.
Armillaria root disease-caused mortality of lychee trees was associated with Desarmillaria caespitosa.
Pines stump could serve as Armillaria inoculum for avocado orchards established in cleared forest areas.
Introduction
The Armillaroid fungi have continued to undergo taxonomic revisions; among them, the incorporation of the genera Guyanagaster T. W. Henkel, M. E. Smith & Aime and Desarmillaria (Herink) R. A. Koch & Aime. The latter contains species without an annulus or ring in the basidiome stipe, which were previously grouped in the genus Armillaria (Fr.) Staude. Armillaria comprises approximately 40 species, some recently described (Elías-Román et al., 2008; Kim et al., 2022; Koch, Wilson, Séne, Henkel, & Aime, 2017).
Desarmillaria comprises three species, D. tabescens (Scop.) R. A. Koch & Aime and D. ectypa (Fr.) R. A. Koch & Aime, both from Eurasia, along with the recently redescribed vicariant, D. caespitosa (Berk.) Antonín, J. E. Stewart & Medel, from the USA and Mexico (Antonín et al., 2021). The recently established taxonomic combination, D. caespitosa (basionym: Lentinus caespitosus Berk.), was previously reported as Clitocybe tabescens (Fr.) Bres. by Cohen (1955) and Desarmillaria tabescens by Miller et al. (2020) causing tree mortality in lychee (Litchi chinensis Sonn.) and peach (Prunus persica [L.] Batsch) orchards, respectively in the southeastern USA. In Veracruz, Mexico, D. caespitosa has been reported as Armillaria tabescens (Scop.) Emel infecting araucaria tree (Araucaria araucana [Molina] K. Koch) in an urban area (Kim et al., 2010); however, D. caespitosa has not been previously documented in association with the lychee trees in the primary lychee-production area in Mexico (Sistema de Información Agroalimentaria y Pesca [SIAP], 2019). The presence of Armillaria spp. has been reported affecting avocado (Persea americana Mill.) orchards in several municipalities in the state of Michoacán, which represents the main avocado-producing area in the world; however, the presence and identification of the Armillaria spp. has not been confirmed in a refereed journal (Hernández-Ramos, Moreno-Velázquez, López-Buenfil, Zelaya-Molina, & Elías-Román, 2018; Michua-Cedillo et al., 2016; Ordas-Ochoa, 2017).
Accurate identification of Armillaria and Desarmillaria species is critical because these species display diverse ecological behaviors and occupy disparate geographic areas. For example, these genera contain species that vary in pathogenicity and virulence (Cleary, van der Kamp, & Morrison, 2012; Kim et al., 2022; Nowakowska et al., 2020); some Armillaria and Desarmillaria species are symbionts of orchids (Gastrodia elata Blume) cultivated for medicinal purposes (Guo, Wang, Xue, Zhao, & Yang, 2016) and other fungi (Polyporus umbellatus [Pers.]) (Xing, Men, & Guo, 2017). Among other ecological functions, one species, A. altimontana Brazee, B. Ortiz, Banik & D. L. Lindner, was reported as a potential biological control agent against a virulent species, A. solidipes Peck. (Warwell et al., 2019).
Because Armillaria and Desarmillaria species associated with tree disease and mortality possess differences in virulence, host range, and climatic adaption (potential geographic range), it especially is important to identity the pathogenic species to develop/apply appropriate disease management practices. Previously, the species of these genera were identified primarily by basidiomata morphology, but basidiomata are ephemeral, produced only sporadically depending on the environmental conditions, and some species may exhibit similar morphology. Biological species defined by interfertility also contributed to the identification process for Armillaria and Desarmillaria species (Anderson & Ullrich, 1979; Korhonen, 1978), but interfertility can be observed among disparate species that have been geographically separated (Heinzelmann et al., 2019; Klopfenstein et al., 2017). In recent decades, DNA sequences have been demonstrated to be especially useful for species identification of these genera (Heinzelmann et al., 2019). The sequences of translation elongation factor 1-alpha (tef1) allow the identification of Armillaria species that cannot be identified by ribosomal DNA, such as internal transcribed spacer (ITS) with the 3’ end of the large subunit (LSU), and the intergenic spacer 1 (IGS1) (3' LSU-IGS1) (Kim, Klopfenstein, Hanna, & McDonald, 2006; Klopfenstein et al., 2017). For the aforementioned reasons, the objective of this study was to identify the Armillaria/Desarmillaria species associated with hosts (lychee, avocado, and pine) with signs and/or symptoms of Armillaria root disease within or in close proximity to orchards of Veracruz and Michoacán, Mexico.
Materials and methods
Collection sites
During October and November 2019, surveys were conducted on a) trees of lychee variety 'Racimo Rojo' (average stem circumference at 40 cm above ground level = 48.5 cm; 15 years after plantation establishment) within orchard #1 (18.659528, -96.920972, 609 m) in Zacatal Chico, Zongolica, Veracruz; b) three orchards of avocado ‘Hass’ variety on rootstocks of the Mexican landrace Persea americana var. drymifolia (Schltdl. & Cham.) S. F. Blake, with trees of 3-3.5 m height and 4-5 years average age since establishment in Michoacán: orchard #2 (19.334982, -102.165026; 1 674 m); orchard #3 (19.34375, -102.17336; 1 667 m) in La Escondida, Uruapan; and orchard #4 (19.384093, -102.18279; 2 115 m) located in Los Lobos, Nuevo Parangaricutiro, and c) a root of pine stump (unidentified Pinus species) in a forest area (19.38106, -102.18420, 2 052 m) adjacent to orchard #4 in Nuevo Parangaricutiro, Michoacán.
Sampling and isolation of Armillaria/Desarmillaria
Recently dead trees or trees with wilt symptoms were selected for sampling. The root system of the trees was inspected and excavated to observe signs (rotten wood, mycelial fans, rhizomorphs, and/or basidiomata) typical of Armillaria root disease (Figure 1). The fungus was isolated from infected roots of trees following the procedure described by Harrington, Worrall, and Baker (1992) in Benomyl-Dichloran-Streptomycin (BDS) medium, and incubated for 3-5 weeks at room temperature (20-26 °C). Subsequently, the fungal isolates were transferred to a nylon filter overlaying 3 % malt-agar medium to obtain abundant mycelial from which DNA was extracted.
Figure 1 Symptoms and signs of Armillaria root disease in lychee (Litchi chinensis) and avocado (Persea americana). a) Avocado orchard with an asymptomatic tree and another symptomatic (chlorosis and decline) tree; b) Sudden mortality of an avocado tree; c) Cortical cracking with dark-colored exudate due to Armillaria mexicana infection near the base of avocado stem; d) Mycelial fan in symptomatic avocado tree crown; e) Rhizomorphs attached to an avocado root; f) Mycelial fan of Desarmillaria caespitosa on the root of a recently dead lychee tree; and g) D. caespitosa basidiomata on lychee tree base.
Tef1 amplification, sequencing, and sequence analyses
PCR amplification and sequencing of tef1 gene followed the protocol of Elías-Román et al. (2018); and DNA sequences were manually edited and aligned with BioEdit 7.1 (Hall, 1999). Polymorphic sites were coded using the IUPAC codes for ambiguous nucleotides. Edited tef1 sequences of isolates obtained from avocado, lychee, and pine trees were compared with sequences available in the NCBI (National Center for Biotechnology Information) database using BLAST nucleotide search and deposited in GenBank. Isolates derived from mycelial fans were deposited at the USDA Forest Service, Rocky Mountain Research Station (RMRS), Forest Sciences Laboratory in Moscow, Idaho, USA.
Results and Discussion
Detection of Armillaria root disease
Eight isolates were obtained from mycelial fans of infected roots. The three isolates (Ver2, Ver4, and Ver5) that were collected from recently dead lychee trees of orchard #1, displayed cottony, whitish growth, without the presence of rhizomorphs in 3% malt-agar medium after approximately 4 weeks of culture. Four other isolates (Mich32, Mich35, Mich51, and Mich52) were obtained from recently dead and symptomatic ‘Hass’ avocado trees grafted on P. americana var. drymifolia rootstocks: The isolates Mich32 and Mich35 were collected from orchards #2 and 3, respectively, in La Escondida, Uruapan, Michoacán; isolates Mich51 and Mich52 were collected in orchard #4 in Los Lobos, Nuevo Parangaricutiro, Michoacán. Mich51 was isolated from an avocado tree with symptoms of decline and cortical cracking, with a dark-colored exudate near the base of the stem (Figures 1a, 1c, and 1d), and other isolates (Mich32, Mich35, and Mich52) were obtained from recently dead trees. Another isolate (Mich4) was obtained from a pine stump in an area adjacent to orchard #4 of avocado. Isolates obtained from avocado trees and pine stump formed crustose mycelium of variable color (light to dark brown) that produced abundant rhizomorphs, and culture exudates that produced a brown stain in the culture medium.
Tef1 sequence analyses
Three isolates (Ver2, Ver4, and Ver5) collected from roots (Figure 1f) of lychee trees from orchard #1 in Zongolica, Veracruz were identified as D. caespitosa based on 1215 bp sequences of tef1 (GenBank accession numbers: MZ851975, MZ851976, and MZ851977 for Ver2, Ver4 and Ver5, respectively), which displayed a very high similarity (99.7 %) with available tef1 sequences in GenBank BLAST that were derived from D. caespitosa isolates, such as GenBank Accession No. MT232068 from isolate OOI-210 obtained from peach (Antonín et al., 2021). Armillaria root disease due to D. caespitosa (reported as Clitocybe tabescens) has been associated with the mortality of lychee trees, and management strategies were subsequently recommended, such as reducing inoculum sources, among other measures (Cohen, 1955). Volk & Burdsall (1995) previously considered C. tabescens as a synonym for A. tabescens; however, the North American vicariant of A. tabescens s. l. is currently recognized as D. caespitosa (Antonín et al., 2021).
The five isolates collected from avocado and pine were identified as A. mexicana R. Elías, Medel, Alvarado, Hanna, Ross-Davis, Kim, & Klopfenstein based on tef1 sequences (GenBank accessions Nos. MZ851978, MZ851979, MZ851980, MZ851981, and MZ851982 for isolated Mich4, Mich32, Mich35, Mich51, and Mich52, respectively). A tef1 sequence length of 1 150 bp was obtained from isolate Mich32, and 1202 bp sequences were obtained from isolates Mich4, Mich35, Mich51, and Mich52. The GenBank nucleotide Blast displayed an extremely high similarity (99.9 %) for tef1 from these five isolates with that of A. mexicana (GenBank Accession No KR061313). This species, first reported as Armillaria sp., was previously found in Zinapécuaro, Michoacán causing mortality of peach and plum (reported as Prunus domestica) trees (Rivas-Valencia et al., 2017), and A. mexicana was also previously found to be pathogenic and more virulent than A. mellea (Vahl) P. Kumm on four Prunus spp. evaluated in greenhouse and field conditions in the State of Mexico (Elías-Román et al., 2019), where it was also previously reported on an oak (Quercus sp.) stump in a forest area (Elías-Román et al., 2018). Other investigations mentioned the presence of A. mexicana on trees of 'Hass' avocado grafted onto Mexican landrace seedling rootstocks; however, tef1 sequences were not deposited in GenBank to allow verification of these previous identifications (Hernández-Ramos et al., 2018; Michua-Cedillo et al., 2016). Of note is that sequences of 3’ LSU-IGS1 of ribosomal DNA in GenBank (Accession Nos. KU378660, KU378658, KU378657, KU378654, KU378655, KU378659) show high similarity with A. mexicana (GenBank Accession No. KR061306); however, those GenBank accessions are not associated with in any publication that discusses their origins. The 3’ LSU-IGS1 region is useful in the identification of A. mexicana (Elías-Román et al., 2013), but it cannot distinguish among some Armillaria species such as A. gallica Marxm. & Romagn., A. sinapina Bérubé & Dessur. and A. cepistipes Velen. (Kim et al., 2006). Additionally, several Armillaria species have very diverse 3’ LSU-IGS1 sequences that could not be aligned unambiguously for phylogenetic analysis.
Climate-change projections suggest that Armillaria root disease will likely increase in areas where the climate will become less suitable for hosts, while remaining suitable for pathogens (Kim et al., 2021; Klopfenstein, Kim, Hanna, Richardson, & Lundquist, 2011). In addition, we consider that incidence and severity of Armillaria root disease could increase in several fruit-growing areas of Mexico due to multiple factors, such as 1) use of substrates contaminated with Armillaria/Desarmillaria propagules for plant propagation in nurseries; 2) change of land use (e.g., replacement of pine-oak forest with fruit orchards) that could promote the spread of pathogenic Armillaria/Desarmillaria species, from previously occupied forested sites; 3) use of trees that are maladapted to the site or become maladapted due to climate change; and/or 4) other biotic and abiotic disturbances that create conditions suitable for Armillaria root disease.
Conclusions
Armillaria root disease in ‘Hass’ avocado trees on Mexican landrace rootstocks Persea americana var. drymifolia in orchards in Michoacán was associated with the presence of Armillaria mexicana. This species was also isolated from a pine tree roots in a forest near avocado orchards, which could represent an inoculum source for infection in avocado orchards established in areas where the natural forest was cleared. In addition, Desarmillaria caespitosa was identified in association with Armillaria root disease of recently dead lychee trees in Zongolica, Veracruz. This study represents the first reports of A. mexicana causing Armillaria root disease on avocado and pine in Michoacán, and D. caespitosa causing Armillaria root disease on lychee in Veracruz. These results add to baseline information on the distribution and host associations of these Armillaria root disease pathogens.
