First Look Into the Ambrosia Beetle-Fungus Symbiosis Present in Commercial Avocado Orchards in Michoacán, Mexico.

Most beetle-fungus symbioses do not represent a threat to agricultural and natural ecosystems; however, a few beetles are able to inoculate healthy hosts with disease-causing fungal symbionts. Here, we report the putative nutritional symbionts associated with five native species of ambrosia beetles colonizing commercial avocado trees in four locations in Michoacán. Knowing which beetles are present in the commercial orchards and the surrounding areas, as well as their fungal associates, is imperative for developing a realistic risk assessment and an effective monitoring system that allows for timely management actions. Phylogenetic analysis revealed five potentially new, previously undescribed species of Raffaelea, and three known species (R. arxi, R. brunnea, R. fusca). The genus Raffaelea was recovered from all the beetle species and across the different locations. Raffaelea lauricola (RL), which causes a deadly vascular fungal disease known as laurel wilt (LW) in Lauraceae species, including avocado, was not recovered. This study points to the imminent danger of native ambrosia beetles spreading RL if the pathogen is introduced to Mexico's avocado orchards or natural areas given that these beetles are associated with Raffaelea species and that lateral transfer of RL among ambrosia beetles in Florida suggests that the likelihood of this phenomenon increases when partners are phylogenetically close. Therefore, this study provides important information about the potential vectors of RL in Mexico and other avocado producing regions. Confirming beetle-fungal identities in these areas is especially important given the serious threat laurel wilt disease represents to the avocado industry in Mexico.

Partnerships Between Ambrosia Beetles and Fungi: Lineage-Specific Promiscuity Among Vectors of the Laurel Wilt Pathogen, Raffaelea lauricola.

Nutritional mutualisms that ambrosia beetles have with fungi are poorly understood. Although these interactions were initially thought to be specific associations with a primary symbiont, there is increasing evidence that some of these fungi are associated with, and move among, multiple beetle partners. We examined culturable fungi recovered from mycangia of ambrosia beetles associated with trees of Persea humilis (silk bay, one site) and P. americana (avocado, six commercial orchards) that were affected by laurel wilt, an invasive disease caused by a symbiont, Raffaelea lauricola, of an Asian ambrosia beetle, Xyleborus glabratus. Fungi were isolated from 20 adult females of X. glabratus from silk bay and 70 each of Xyleborus affinis, Xyleborus bispinatus, Xyleborus volvulus, Xyleborinus saxesenii, and Xylosandrus crassiusculus from avocado. With partial sequences of ribosomal (LSU and SSU) and nuclear (ß-tubulin) genes, one to several operational taxonomic units (OTUs) of fungi were identified in assayed individuals. Distinct populations of fungi were recovered from each of the examined beetle species. Raffaelea lauricola was present in all beetles except X. saxesenii and X. crassiusculus, and Raffaelea spp. predominated in Xyleborus spp. Raffaelea arxii, R. subalba, and R. subfusca were present in more than a single species of Xyleborus, and R. arxii was the most abundant symbiont in both X. affinis and X. volvulus. Raffaelea aguacate was detected for the first time in an ambrosia beetle (X. bispinatus). Yeasts (Ascomycota, Saccharomycotina) were found consistently in the mycangia of the examined beetles, and distinct, putatively co-adapted populations of these fungi were associated with each beetle species. Greater understandings are needed for how mycangia in ambrosia beetles interact with fungi, including yeasts which play currently underresearched roles in these insects.

First Report of Fusarium oxysporum f. sp. cubense Tropical Race 4 Associated with Panama Disease of Banana outside Southeast Asia.

Fusarium wilt or Panama disease of banana, caused by Fusarium oxysporum f. sp. cubense (Foc), is among the most destructive plant diseases (3). Race 1 ravaged 'Gros Michel'-based export trades until the cultivar was replaced by resistant Cavendish cultivars. However, a new variant of Foc, tropical race 4 (TR4), was identified in Southeast Asia in 1992 and has spread throughout the region (3). Cavendish clones, which are most important in subsistence and export production, are among the wide range of cultivars that are affected, and there is a huge concern that TR4 will further disseminate in Africa since its presence was announced in November 2013 and move into Latin America, thereby threatening other vital banana-growing regions. In Jordan, Cavendish bananas are produced on 1,000 to 1,500 ha in the Jordan Valley (32°N, 35.5°E). In 2006, symptoms of Fusarium wilt were observed and sampled for the isolation of Foc. On half-strength PDA amended with 100-ppm streptomycin sulfate, pale salmon-colored colonies with floccose mycelia developed consistently from surface-disinfested xylem. Single microconidia from these colonies were transferred to half-strength PDA, and conidia and mycelia from these monospore colonies were stored at -80°C in 15% glycerol. On banana leaf agar (Co60-irradiated leaf tissue on water agar), isolates resembled F. oxysporum phenotypically by producing infrequent three- to five-celled macroconidia, copious, usually aseptate microconida on monophialides, and terminal and intercalary chlamydospores after 2 weeks (2). With nitrate-nonutilizing (nit) mutants and testers for different vegetative compatibility groups (VCGs), each of seven examined monospore isolates were placed in VCG 01213, which contains only strains of TR4 (3). Total DNA was extracted from six isolates and PCR analyses, which confirmed their identity as TR4 (1). Subsequently, one of the isolates (JV11) was analyzed for pathogenicity. Inoculum production and inoculation were according to (1) by dipping (30 min) root-wounded 10-week-old plants of the Cavendish cv. Grand Naine in 2 liters of spore suspension (1.0 × 106 spores/ml). Inoculated plants were then placed in sand in 3-liter pots under 28°C, 70% relative humidity, and a 16/8-h light/darkness photoperiod. Sets of three plants were each treated with either JV11 or two TR4 controls (isolate II-5 and a strain isolated from an affected Cavendish plant in Mindanao, Philippines, both of which were diagnosed as TR4 by PCR and pathogenicity analyses). Control sets were either treated with race 1 originating from Cruz das Almas, Bahia, Brazil (1), or water. After 2 weeks, plants inoculated with JV11 and TR4 controls produced typical symptoms of Fusarium wilt. After 4 weeks, tissue was collected from all plants and plated on Komada's medium. TR4 was directly confirmed by PCR (1), either directly from symptomatic plants (JV11 and TR4 controls), or from isolates that were recovered from these plants. Nothing was re-isolated from race 1 inoculated plants and water controls, which remained asymptomatic. This is the first report of TR4 affecting Cavendish outside Southeast Asia, is its northernmost outbreak, and represents a dangerous expansion of this destructive race. Currently, 80% of the Jordan Valley production area is affected by Fusarium wilt, and 20 to 80% of the plants are affected in different farms. References: (1) M. A. Dita et al. Plant Pathol. 59:348, 2010. (2) J. F. Leslie and B. A. Summerell. The Fusarium Lab Manual. Blackwell, Ames, 2006. (3) R. C. Ploetz. Phytopathology 96:653, 2006.

First Report of Gulf Licaria, Licaria trianda, as a Suscept of Laurel Wilt.

Gulf licaria, Licaria trianda (Sw.) Kosterm., is a federally endangered member of the Lauraceae plant family in Miami-Dade County, Florida. It was never common in the area, and urban development has extirpated it from most of its former range; as of 2001, fewer than 10 trees remained in a single, remnant habitat in the continental United States, Simpson Park (25°45'31″N, 80°11'46″W) (2). Laurel wilt, caused by the fungus Raffaelea lauricola T. C. Harr., Fraedrich & Aghayeva, has recently devastated members of the Lauraceae in the southeastern United States, most notably redbay, Persea borbonia (1). As R. lauricola and its vector, the redbay ambrosia beetle Xyleborus glabratus, have spread in the region, an increasing number of taxa in this plant family have been affected by this disease (1). In 2012, seedlings of gulf licaria and redbay were obtained from local nurseries; they were grown in 30 liter pots, 1.3 m tall, had stems 3 cm in diameter 20 cm above the soil line, and were maintained with standard watering and fertilization practices. In two pathogenicity experiments on July 6 and September 25, 2012, three plants each of gulf licaria and redbay were inoculated with an isolate of R. lauricola, RL4, as described in previous experiments (3), and two plants each were mock inoculated (water control). RL4 is deposited as CBS 127349 at the Centraalbureau voor Schimmelcultures (CBS Fungal Biodiversity Centre, Utrecht, The Netherlands), and a SSU rDNA sequence for it is deposited in GenBank under Accession No. HM446155. Beginning 2 weeks after inoculation, plants were rated on a weekly basis for the development of external symptoms, on a subjective 1 (no symptoms) to 10 (dead) scale (3). After 5 weeks, inoculated plants of redbay in each experiment (positive control) had died after first developing symptoms of wilt and necrotic foliage that are typical for this disease (1). In contrast, inoculated plants of gulf licaria developed severe symptoms by the time experiments were terminated 6 and 11 weeks after inoculation; chlorosis developed on some of the leaves of all plants and these eventually abscised (mean external severities of 7.3 and 6.5, respectively), but plants did not die. Brown to greyish discoloration of sapwood developed in all inoculated plants, and the pathogen was recovered from symptomatic sapwood on CSMA (3). No symptoms developed on mock inoculated plants and the pathogen was not recovered from them. It is concluded that gulf licaria is susceptible to laurel wilt, but that it is apparently less susceptible than redbay. Whether X. glabratus is attracted to, or will bore into, gulf licaria is not known, but will play a significant role in the extent to which this rare tree is affected by laurel wilt. References: (1) S. W. Fraedrich et al. Plant Dis. 92:215, 2008. (2) G. D. Gann et al. Rare Plants of South Florida: Their History, Conservation, and Restoration. Institute for Regional Conservation, Miami, 2002. (3) R. C. Ploetz et al. Plant Pathol. 61:801, 2012.