Akif Eskalen

Host Range of Fusarium Dieback and Its Ambrosia Beetle (Coleoptera: Scolytinae) Vector in Southern California

The polyphagous shot hole borer (PSHB) is an invasive ambrosia beetle that forms a symbiosis with a new, as-yet-undescribed Fusarium sp., together causing Fusarium dieback on avocado and other host plants in California and Israel. In California, PSHB was first reported on black locust in 2003 but there were no records of fungal damage until 2012, when a Fusarium sp. was recovered from the tissues of several backyard avocado trees infested with PSHB in Los Angeles County. The aim of this study was to determine the plant host range of the beetle-fungus complex in two heavily infested botanical gardens in Los Angeles County. Of the 335 tree species observed, 207 (62%), representing 58 plant families, showed signs and symptoms consistent with attack by PSHB. The Fusarium sp. was recovered from 54% of the plant species attacked by PSHB, indicated by the presence of the Fusarium sp. at least at the site of the entry hole. Trees attacked by PSHB included 11 species of California natives, 13 agriculturally important species, and many common street trees. Survey results also revealed 19 tree species that function as reproductive hosts for PSHB. Additionally, approximately a quarter of all tree individuals planted along the streets of southern California belong to a species classified as a reproductive host. These data suggest the beetle-disease complex potentially may establish in a variety of plant communities locally and worldwide.

An inordinate fondness for Fusarium: Phylogenetic diversity of fusaria cultivated by ambrosia beetles in the genus Euwallacea on avocado and other plant hosts

Ambrosia beetle fungiculture represents one of the most ecologically and evolutionarily successful symbioses, as evidenced by the 11 independent origins and 3500 species of ambrosia beetles. Here we document the evolution of a clade within Fusarium associated with ambrosia beetles in the genus Euwallacea (Coleoptera: Scolytinae). Ambrosia Fusarium Clade (AFC) symbionts are unusual in that some are plant pathogens that cause significant damage in naïve natural and cultivated ecosystems, and currently threaten avocado production in the United States, Israel and Australia. Most AFC fusaria produce unusual clavate macroconidia that serve as a putative food source for their insect mutualists. AFC symbionts were abundant in the heads of four Euwallacea spp., which suggests that they are transported within and from the natal gallery in mandibular mycangia. In a four-locus phylogenetic analysis, the AFC was resolved in a strongly supported monophyletic group within the previously described Clade 3 of the Fusarium solani species complex (FSSC). Divergence-time estimates place the origin of the AFC in the early Miocene ∼21.2 Mya, which coincides with the hypothesized adaptive radiation of the Xyleborini. Two strongly supported clades within the AFC (Clades A and B) were identified that include nine species lineages associated with ambrosia beetles, eight with Euwallacea spp. and one reportedly with Xyleborus ferrugineus, and two lineages with no known beetle association. More derived lineages within the AFC showed fixation of the clavate (club-shaped) macroconidial trait, while basal lineages showed a mix of clavate and more typical fusiform macroconidia. AFC lineages consisted mostly of genetically identical individuals associated with specific insect hosts in defined geographic locations, with at least three interspecific hybridization events inferred based on discordant placement in individual gene genealogies and detection of recombinant loci. Overall, these data are consistent with a strong evolutionary trend toward obligate symbiosis coupled with secondary contact and interspecific hybridization.

Susceptibility of Grapevine Pruning Wounds and Symptom Development in Response to Infection by Phaeoacremonium aleophilum and Phaeomoniella chlamydospora

Fresh to 10-month-old pruning wounds on grapevine cvs. Thompson Seedless and Cabernet Sauvignon were inoculated with Phaeoacremonium aleophilum or Phaeomoniella chlamydospora at approximately 1 × 106 spores/ml. Successful infection was determined by isolation of the pathogen from necrotic margins of cankers or from vascular discoloration assessed 4 months after each inoculation date. Disease incidence decreased as the length of time between pruning and inoculation increased; however, wounds remained susceptible for up to 4 months. Vascular discoloration was significantly less extensive in the noninoculated control than in inoculated spurs. Reduction of shoot length in both cultivars varied depending on pruning wound age at the time of inoculation. In a separate study, inoculation of fresh pruning wounds of Cabernet Sauvignon resulted in successful infection regardless of the time of pruning from February to December.

Association of spores of Phaeomoniella chlamydospora, Phaeoacremonium inflatipes, and Pm. aleophilum with grapevine cordons in California.

Summary. Esca (black measles) of grapevine has long been known to occur wherever grapes are grown. Phaeomoniella chlamydospora and two species of Phaeoacremonium, Pm. inflatipes and Pm. aleophilum, have been associated with esca and Petri grapevine decline in major production regions of California. Though present in symptomatic grapevines and capable of causing foliar symptoms of esca, Phaeomoniella chlamydospora does not cause the typical symptoms on fruit. However, trapping studies showed that spores of Pa. chlamydospora, Pm. inflatipes and Pm. aleophilum were captured throughout the year in vineyards ranging from the north California coast to the southern San Joaquin Valley. They can be considered airborne fungi capable of being water-splashed by pruning or other wounds during part of their biological cycle. Trapping of spores coincided with rainfall events for Pa. chlamydospora and Pm. inflatipes, and to a lesser degree for Pm. aleophilum. However, this last species was trapped during periods of time when rainfall did not occur and was trapped longer into the summer.

Root diseases of grapevines in California and their control

Root rots of grapevine in California are caused by several soilborne pathogens. While root rot in vineyards in general has been considered to be a relative minor problem, nearly all root diseases have increased in incidence and severity since the introduction of rootstocks resistant to Phylloxera. Pathogens such as Phytophthora spp. and Armillaria mellea have been known to occur for many years. However, seldom did root rot become a serious problem in vineyards. New diseases such as Petri disease caused by Phaeomoniella chlamydospora, vine decline caused by Phaeoacremonium spp. and black foot caused by Cylindrocarpon destructans have only recently been shown to occur in California. Wilt caused by Verticillium dahliae has become more prevalent in nearly all grape growing regions in California. Though losses due to actual root disease have been minor over the past 10 years, losses due to vineyard reestablishment have been large.

Discordant phylogenies suggest repeated host shifts in the Fusarium-Euwallacea ambrosia beetle mutualism.

The mutualism between xyleborine beetles in the genus Euwallacea (Coleoptera: Curculionidae: Scolytinae) and members of the Ambrosia Fusarium Clade (AFC) represents one of 11 known evolutionary origins of fungiculture by ambrosia beetles. Female Euwallacea beetles transport fusarial symbionts in paired mandibular mycangia from their natal gallery to woody hosts where they are cultivated in galleries as a source of food. Native to Asia, several exotic Euwallacea species were introduced into the United States and Israel within the past two decades and they now threaten urban landscapes, forests and avocado production. To assess species limits and to date the evolutionary diversification of the mutualists, we reconstructed the evolutionary histories of key representatives of the Fusarium and Euwallacea clades using maximum parsimony and maximum likelihood methods. Twelve species-level lineages, termed AF 1-12, were identified within the monophyletic AFC and seven among the Fusarium-farming Euwallacea. Bayesian diversification-time estimates placed the origin of the Euwallacea-Fusarium mutualism near the Oligocene-Miocene boundary ∼19-24 Mya. Most Euwallacea spp. appear to be associated with one species of Fusarium, but two species farmed two closely related fusaria. Euwallacea sp. #2 in Miami-Dade County, Florida cultivated Fusarium spp. AF-6 and AF-8 on avocado, and Euwallacea sp. #4 farmed Fusarium ambrosium AF-1 and Fusarium sp. AF-11 on Chinese tea in Sri Lanka. Cophylogenetic analyses indicated that the Euwallacea and Fusarium phylogenies were largely incongruent, apparently due to the beetles switching fusarial symbionts (i.e., host shifts) at least five times during the evolution of this mutualism. Three cospeciation events between Euwallacea and their AFC symbionts were detected, but randomization tests failed to reject the null hypothesis that the putative parallel cladogenesis is a stochastic pattern. Lastly, two collections of Euwallacea sp. #2 from Miami-Dade County, Florida shared an identical cytochrome oxidase subunit 1 (CO1) allele with Euwallacea validus, suggesting introgressive hybridization between these species and/or pseudogenous nature of this marker. Results of the present study highlight the importance of understanding the potential for and frequency of host-switching between Euwallacea and members of the AFC, and that these shifts may bring together more aggressive and virulent combinations of these invasive mutualists.

First Report of a Fusarium sp. and Its Vector Tea Shot Hole Borer (Euwallacea fornicatus) Causing Fusarium Dieback on Avocado in California

Per capita consumption of avocado in the United States has nearly doubled between 2000 and 2010. The California avocado industry supplies almost 40% of U.S. demand and the remaining 60% is supplied by imports from Latin America and New Zealand. The Tea Shot Hole Borer (TSHB) is an ambrosia beetle from Asia that forms a symbiosis with a new, yet undescribed Fusarium sp. and is a serious problem for the Israeli avocado industry (3). The beetle also causes severe damage on the branches of tea (Camelia sinensis) in Sri Lanka and India (1). In California, TSHB was first reported on black locust (Robinia pseudoacacia) in 2003, but there are no records of fungal damage (4). In 2012, nine backyard avocado trees (cvs. Hass, Bacon, Fuerte, and Nabal) exhibiting branch dieback were observed throughout the residential neighborhoods of South Gate, Downey, and Pico Rivera in Los Angeles County. Upon inspection, symptoms of white powdery exudate, either dry or surrounded by wet discoloration of the outer bark in association with a single beetle exit hole, were found on the trunk and main branches of the tree. Examination of the cortex and wood under the exit hole revealed brown discolored necrosis. The TSHB was also found within galleries that were 1 to 4 cm long going against the grain. Symptomatic cortex and sapwood tissues were plated onto potato dextrose agar amended with 0.01% tetracycline (PDA-tet). The TSHB was dissected and plated onto PDA-tet after surface disinfestation following methods described by Kajimura and Hijii (2). After 5 days of incubation at room temperature, regular fungal colonies with aerial mycelia and reddish brown margins were produced. Single spore isolations were used to establish pure culture of the fungus. Fifty conidia were hyaline, clavate with a rounded apex, and initially aseptate (4.1 to 12.0 × 2.4 to 4.1 μm) becoming one- to three-septate (7.6 to 15.1 × 2.8 to 4.5 μm, 9.2 to 17.2 × 3.4 to 4.8 μm, and 13.5 to 17.6 × 4.3 to 4.7 μm, respectively). Identity of the fungal isolates was determined by amplification of the rDNA genes with primers ITS4/5 and EF1/2, respectively. Sequences were deposited into GenBank under Accession Nos. JQ723753, JQ723760, JQ723756, and JQ723763. A BLASTn search revealed 100% similarity to Fusarium sp. (Accession Nos. JQ038020 and JQ038013). Detached green shoots of healthy 1-year-old avocado were wounded to a depth of 1 to 2 mm and 5-mm mycelial plugs from 5-day-old cultures (UCR 1781 and UCR 1837) were placed mycelial side down onto the freshly wounded surfaces and then wrapped with Parafilm. Control shoots were inoculated with sterile agar plugs and five replicates per treatment were used. Shoots were incubated at 25 ± 1°C in moist chambers for 3 weeks. Lesions were observed on all inoculated shoots except for the control. Mean lesion lengths were 10.7 and 12.8 cm for UCR1781 and UCR1837, respectively, and were significantly different (P ≤ 0.05) from the control. Both isolates were reisolated from 100% of symptomatic tissues of inoculated shoots to complete Kochs postulates. This experiment was conducted twice and similar results were obtained. To our knowledge, this is the first report of Fusarium sp. and its vector E. fornicatus causing Fusarium dieback on Avocado in California. References: (1) W. Danthanarayana. Tea Quarterly 39:61, 1968. (2) H. Kajimura and N. Hijii. Ecol. Res. 7:107, 1992; (3) Mendel et al., Phytoparasitica, DOI 10.1007/s12600-012-0223-7, 2012. (4) R. J. Rabaglia. Annals Entomol. Soc. Amer. 99:1034, 2006.

Fusarium euwallaceae sp. nov.—a symbiotic fungus of Euwallacea sp., an invasive ambrosia beetle in Israel and California

The invasive Asian ambrosia beetle Euwallacea sp. (Coleoptera, Scolytinae, Xyleborini) and a novel Fusarium sp. that it farms in its galleries as a source of nutrition causes serious damage to more than 20 species of live trees and pose a serious threat to avocado production (Persea americana) in Israel and California. Adult female beetles are equipped with mandibular mycangia in which its fungal symbiont is transported within and from the natal galleries. Damage caused to the xylem is associated with disease symptoms that include sugar or gum exudates, dieback, wilt and ultimately host tree mortality. In 2012 the beetle was recorded on more than 200 and 20 different urban landscape species in southern California and Israel respectively. Euwallacea sp. and its symbiont are closely related to the tea shot-hole borer (E. fornicatus) and its obligate symbiont, F. ambrosium occurring in Sri Lanka and India. To distinguish these beetles, hereafter the unnamed xyleborine in Israel and California will be referred to as Euwallacea sp. IS/CA. Both fusaria exhibit distinctive ecologies and produce clavate macroconidia, which we think might represent an adaption to the species-specific beetle partner. Both fusaria comprise a genealogically exclusive lineage within Clade 3 of the Fusarium solani species complex (FSSC) that can be differentiated with arbitrarily primed PCR. Currently these fusaria can be distinguished only phenotypically by the abundant production of blue to brownish macroconidia in the symbiont of Euwallacea sp. IS/CA and their rarity or absence in F. ambrosium. We speculate that obligate symbiosis of Euwallacea and Fusarium, might have driven ecological speciation in these mutualists. Thus, the purpose of this paper is to describe and illustrate the novel, economically destructive avocado pathogen as Fusarium euwallaceae sp. nov. S. Freeman et al.

Botryosphaeriaceae Species Associated with Avocado Branch Cankers in California

Members of the family Botryosphaeriaceae cause branch cankers and dieback on California avocado trees. More intensive pruning, a practice associated with high-density planting that is becoming more common in the California avocado industry, may increase the occurrence of branch canker. This study was undertaken to identify and characterize the Botryosphaeriaceae spp. involved in the branch canker disease complex in order to develop future management strategies. From 2008 to 2009, branch cankers were sampled from four or five trees from each of eight avocado groves in five California counties. Six Botryosphaeriaceae spp. were identified based on morphology as well as phylogenetic analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) and a partial sequence of the β-tubulin gene. These six species included Neofusicoccum australe, N. luteum, N. parvum, an unknown Neofusicoccum sp., Fusicoccum aesculi, and Dothiorella iberica. Members of the Botryosphaeriaceae were isolated from all avocado-growing regions sampled in California; however, incidence and distribution of species varied. This report is the first description of the isolation of D. iberica from avocado branch cankers in California.

Teleomorph Formation of Phaeoacremonium aleophilum, Cause of Esca and Grapevine Decline in California

Phaeoacremonium is a recently described genus of the hyphomycetes and includes species associated with grapevine (Vitis vinifera) declines worldwide. Spores of Phaeoacremonium spp. have been trapped in infested vineyards, but neither asexual nor sexual fruiting structures have been observed in the field. Mating studies were carried out to determine if California P. aleophilum isolates are capable of forming a teleomorph in vitro. Sterilized grapevine shavings were placed on the surface of water agar plates with pairs of different California isolates of P. aleophilum, an isolate from the holotype of P. aleophilum, plus other related Phaeoacremonium spp. After approximately 28 to 35 days, perithecia were seen forming on wood chips and agar of many pairings. Upon maturation, fertile perithecia had gelatinous droplets of ascospores oozing from their ostioles. Successful crosses, resulting in mature perithecia, corresponded to a heterothallic mating type system. When F1 progeny were backcrossed with their parents, heterothallism was confirmed. Molecular analyses of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA from ascospore progeny and perithecia confirmed that these perithecia were the teleomorph of P. aleophilum, Togninia minima. Furthermore, 4 months after moist incubating grapevine pieces from naturally infected vineyards, mature perithecia of T. minima could be seen forming on the xylem and pith tissues, suggesting both mating types occur on the same vine.