First report of Fusarium euwallaceae on avocado trees in Palestine

Abstract

A new die back symptoms in many avocado orchards had been reported in Palestine. The disease is associated with the Ambrosia beetle Euwallacea fornicatus. Stem samples from infected avocado trees with obvious symptoms were collected from different regions in Palestine. Stem cuttings and dissected adult and galleries of the insect were placed on potato dextrose agar media and incubated for 5–7?days at 25?°C. PCR amplification using EF1/2 specific primers was performed to identify the isolated fungus. The resulting PCR products were sequenced. BLASTn search showed 99% similarity with Fusarium euwallaceae (Accession Nos. JX891785.1, JQ723763.1, JQ723762.1 and JQ723761.1). The isolated fungus was identified as F. euwallaceae (Genbank accession no. MK054177).     

Obligate feed requirement of Fusarium sp. nov., an avocado wilting agent, by the ambrosia beetle Euwallacea aff. fornicata

The ambrosia beetle, Euwallacea aff. fornicata Eichhoff was first recorded in Israel in 2009. The symbiotic fungus Fusarium sp. nov., carried in the mandibular mycangia of the beetle, is responsible for typical wilt and dieback symptoms inflicted on avocado (Persea americana Miller) trees. The beetle-fungus complex has become a serious threat to the future of the avocado industry in Israel and elsewhere. When reared on Petri dishes, inoculated with 7-day-old cultures of the symbiotic Fusarium sp. nov., the beetle successfully completed its lifecycle and developed from egg to fertile adults in approximately 60 days. Galleries that were produced in the PDA medium by the adults, resembled those excavated in host plant xylem under natural host colonization conditions. Euwallacea aff. fornicata from avocado in Israel was not able to survive when fed with F. ambrosium but resulted in approximately 25 % mortality when fed on F. solani; both isolates originated from infected tea. Likewise, the larvae of E. fornicatus from tea in Sri Lanka, were not able to survive or complete their lifecycle when supplied with a feed of the Fusarium sp. nov. isolated from avocado in Israel. Isolates of two other Fusaria, F. mangiferae from mango and F. oxysporum f. sp. melonis from melon, were not able to support development or survival of the beetle larvae from avocado from Israel, using the same Petri dish rearing method. This indicates that the Fusarium sp. nov. isolate from avocado is obligately required for the survival and development of Euwallacea aff. fornicata currently occurring in Israel, affecting this crop and additional hosts. The usefulness of the Petri dish assay to study the interactions between ambrosia beetles and their fungal symbionts is discussed.     

Differences among avocado cultivars in susceptibility to polyphagous shot hole borer (Euwallacea spec.)

Polyphagous shot hole borer (PSHB) (Euwallacea spec.; Coleoptera: Curculionidae: Scolytinae) has recently invaded southern California, USA. Along with its associated fungi, the beetle causes branch dieback and tree mortality in more than 200 host tree species, including avocado (Persea americana Mill., Lauraceae) and many important native, urban landscape, and nursery stock trees. As PSHB spreads into the avocado‐growing regions of southern California, there is a pressing need for more information for effective management of the insect/fungus complex. The objective of this study was to examine avocado cultivars to see whether there were differences in susceptibility to attack and gallery development from PSHB. We conducted choice and no‐choice trials with cut branches in the laboratory, artificially infested avocado saplings with PSHB, and also surveyed attack rates for two cultivars of field‐grown avocado subject to natural attack by PSHB. Laboratory and field trials showed similar patterns for preference among avocado cultivars indicated by beetle attack rates and gallery formation. Among the common commercial cultivars, Fuerte, Gwen, and Bacon most often had lower attack rates and lower rates of gallery formation. Zutano most frequently indicated high attack rates and much gallery formation, indicating that it may be the most susceptible to PHSB of the commercial cultivars.     

Fungal associates of the tree-killing bark beetle,Ips typographus,vary in virulence,ability to degrade conifer phenolics and influence bark beetle tunneling behavior

The bark beetle Ips typographus carries numerous fungi that could be assisting the beetle in colonizing live Norway spruce (Picea abies) trees. Phenolic defenses in spruce phloem are degraded by the beetle's major tree-killing fungus Endoconidiophora polonica, but it is unknown if other beetle associates can also catabolize these compounds. We compared the ability of five fungi commonly associated with I. typographus to degrade phenolic compounds in Norway spruce phloem. Grosmannia penicillata and Grosmannia europhioides were able to degrade stilbenes and flavonoids faster than E. polonica and grow on minimal growth medium with spruce bark constituents as the only nutrients. Furthermore, beetles avoided medium amended with phenolics but marginally preferred medium colonized by fungi. Taken together our results show that different bark beetle-associated fungi have complementary roles in degrading host metabolites and thus might improve this insect's persistence in well defended host tissues.     

Broadscale Specificity in a Bark Beetle–Fungal Symbiosis: a Spatio-temporal Analysis of the Mycangial Fungi of the Western Pine Beetle

Whether and how mutualisms are maintained through ecological and evolutionary time is a seldom studied aspect of bark beetle–fungal symbioses. All bark beetles are associated with fungi and some species have evolved structures for transporting their symbiotic partners. However, the fungal assemblages and specificity in these symbioses are not well known. To determine the distribution of fungi associated with the mycangia of the western pine beetle (Dendroctonus brevicomis), we collected beetles from across the insect’s geographic range including multiple genetically distinct populations. Two fungi, Entomocorticium sp. B and Ceratocystiopsis brevicomi, were isolated from the mycangia of beetles from all locations. Repeated sampling at two sites in Montana found that Entomocorticium sp. B was the most prevalent fungus throughout the beetle’s flight season, and that females carrying that fungus were on average larger than females carrying C. brevicomi. We present evidence that throughout the flight season, over broad geographic distances, and among genetically distinct populations of beetle, the western pine beetle is associated with the same two species of fungi. In addition, we provide evidence that one fungal species is associated with larger adult beetles and therefore might provide greater benefit during beetle development. The importance and maintenance of this bark beetle–fungus interaction is discussed.     

Entomopathogenic fungi as biological control agents for the vector of the laurel wilt disease,the redbay ambrosia beetle,Xyleborus glabratus (Coleoptera: Curculionidae)

The redbay ambrosia beetle (RAB), Xyleborus glabratus, is a wood-boring insect that vectors the fungal pathogen, Raffaelea lauricola, which causes laurel wilt, a lethal disease of avocado. The objective of this study was to determine the susceptibility of RAB to infection and subsequent death by exposure to three commercial strains of entomopathogenic fungi [two strains of Isaria fumosorosea (Ifr 3581 and PFR), and strain GHA of Beauveria bassiana]. RAB females were dipped in fungal spore solutions and their median survivorship times (MST) determined. Contact with any of the biopesticides resulted in death of all RAB females. MSTs of RAB females ranged from 3 days (B. bassiana) to 5 days (I. fumosorosea PFR). B. bassiana killed RAB females faster, followed by Ifr 3581 and PFR. RAB females dipped in B. bassiana suspensions had the highest number of viable spores attached to their bodies, followed by Ifr 3581. Beetles dipped in PFR suspension had significantly less viable spores attached to their bodies. No significant differences were observed in the mortality of beetles exposed to entomopathogenic fungi by dipping in a fungal suspension or walking on treated avocado bolts. Beetles bored into the logs and constructed galleries, but they were found dead inside the galleries a few days after exposure to the entomopathogens. Entomopathogenic fungal infection in dead beetles was confirmed through molecular techniques. This is the first study to demonstrate that entomopathogenic fungi are potential biological control agents against RAB.     

Experimental evidence of bark beetle adaptation to a fungal symbiont

The importance of symbiotic microbes to insects cannot be overstated; however, we have a poor understanding of the evolutionary processes that shape most insect–microbe interactions. Many bark beetle (Coleoptera: Curculionidae, Scolytinae) species are involved in what have been described as obligate mutualisms with symbiotic fungi. Beetles benefit through supplementing their nutrient‐poor diet with fungi and the fungi benefit through gaining transportation to resources. However, only a few beetle–fungal symbioses have been experimentally manipulated to test whether the relationship is obligate. Furthermore, none have tested for adaptation of beetles to their specific symbionts, one of the requirements for coevolution. We experimentally manipulated the western pine beetle–fungus symbiosis to determine whether the beetle is obligately dependent upon fungi and to test for fine‐scale adaptation of the beetle to one of its symbiotic fungi, Entomocorticium sp. B. We reared beetles from a single population with either a natal isolate of E. sp. B (isolated from the same population from which the beetles originated), a non‐natal isolate (a genetically divergent isolate from a geographically distant beetle population), or with no fungi. We found that fungi were crucial for the successful development of western pine beetles. We also found no significant difference in the effects of the natal and non‐natal isolate on beetle fitness parameters. However, brood adult beetles failed to incorporate the non‐natal fungus into their fungal transport structure (mycangium) indicating adaption by the beetle to particular genotypes of symbiotic fungi. Our results suggest that beetle–fungus mutualisms and symbiont fidelity may be maintained via an undescribed recognition mechanism of the beetles for particular symbionts that may promote particular associations through time.     

Monitoring the decline in AM fungus populations and efficacy during a long term bare fallow

Producing nonmycorrhizal plants in the field is a challenge due to the ubiquitous distribution of arbuscular mycorrhizal [AM] fungi and impacts of chemical treatments upon nontarget organisms. A field plot was covered with ground cover fabric to prohibit plant growth and take advantage of the obligate symbiotic nature of AM fungi to selectively starve and remove them from the soil microbiological community. The decline in the AM fungus population was monitored through spore counts and most probable number bioassays. Response to inoculation experiments were conducted to contrast the response of Allium porrum L. to inoculation with in vitro produced spores of Glomus intraradices Schenck and Smith when plants were grown in the AM fungus-depleted soil vs. soil from an adjacent, cropped plot. Data indicated a strongly diminished, yet still viable population of AM fungi after 39 months of bare fallow. Plants grown in cropped soil showed no growth response nor increase in percentage root length colonized as a result of inoculation, while the response to inoculation of plants grown in the covered soil increased as the population of AM fungi declined below 1 propagule cm^?3.     

A mid-Cretaceous ambrosia fungus, Paleoambrosia entomophila gen. nov. et sp. nov. (Ascomycota: Ophiostomatales) in Burmese (Myanmar) amber,and evidence for a femoral mycangium

An ambrosia fungus is described from filamentous sporodochia adjacent to a wood–boring ambrosia beetle (Coleoptera: Curculionidae: Platypodinae) in mid-Cretaceous Burmese amber. Yeast-like propagules and hyphal fragments of Paleoambrosia entomophila gen. nov. et sp. nov. occur in glandular sac mycangia located inside the femur of the beetle. This is the first record of a fossil ambrosia fungus, showing that symbiotic associations between wood–boring insects and ectosymbiotic fungi date back some 100 million years ago. The present finding moves the origin of fungus-growing by insects from the Oligocene to the mid-Cretaceous and suggests a Gondwanan origin.     

Beetles provide directed dispersal of viable spores of a keystone wood decay fungus

Wood decay fungi are considered to be dispersed by wind, but dispersal by animals may also be important, and more so in managed forests where dead wood is scarce. We investigated whether beetles could disperse spores of the keystone species Fomitopsis pinicola. Beetles were collected on sporocarps and newly felled spruce logs, a favourable habitat for spore deposition. Viable spores (and successful germination) of F. pinicola were detected by dikaryotization of monokaryotic bait mycelium from beetle samples. Viable spores were on the exoskeleton and in the faeces of all beetles collected from sporulating sporocarps. On fresh spruce logs, nine beetle species transported viable spores, of which several bore into the bark. Our results demonstrate that beetles can provide directed dispersal of wood decay fungi. Potentially, it could contribute to a higher persistence of some species in fragmented forests where spore deposition by wind on dead wood is less likely.     

Relative Tolerance of Selected Soybean Cultivars to Hoplolaimus columbus and Possible Effects of Soil Temperature

Eleven soybean [Glycine max (L.) Merr.] cultivars resistant to one or more plant-parasitic nematodes, and one resistant to the Mexican bean beetle (Epilachna varivestis Muls.), were tested for susceptibility to Hoplolaimus columbus. All cultivars were parasitized, but nematode reproduction varied. ''Pickett-71'' was the most susceptible host among the cultivars tested. ''Dyer'' and three ''P.I. cultivars'' were most tolerant when yield /plant and total yield were compared for fumigated and unfumigated plots, even though their yield potential was low. ''Hardee,'' ''Coker 4504,'' ''W-4,'' ''D71-9257,'' and ''ED-371'' appeared tolerant throughout the growing season and yielded well in unfumigated soil. Infection and reproduction of H. columbus in ''Forrest'' soybean roots were greater at 30 ± 1 C than at 20 or 25 ± 1 C. Plant height and root weight varied with the soil treatments.     

Ambrosia fungi in the insect-fungi symbiosis in relation to cork oak decline

Ambrosia fungi live associated with beetles (Scolytidae and Platypodidae) in host trees and act as a food source for the insects. The symbiotic relation is important to the colonizing strategies of host trees by beetles. Ambrosia fungi are dimorphic: they grow as ambrosial form and as mycelium. The fungi are highly specialized, adapted to a specific beetle and to the biotope where they both live. In addition other fungi have been found such as tree pathogenic fungi that may play a role in insects host colonization success. Saprophytic fungi are also present in insects galleries. These may decompose cellulose and/or be antagonistic to other less beneficial fungi. This paper summarizes the importance of ambrosia fungi and the interaction with insects and hosts. The possibility of the transport of pathogenic fungi by Platypus cylindrus to cork oak thus contributing for its decline is discussed.     

Xylanase and xylosidase activities in avocado fruit

The activities of xylanase and xylosidase were demonstrated in mature avocado (Persea americana Mill.) fruits from different cultivars. When monitored on the day of harvest during the season at 1-month intervals, xylanase activity decreased and xylosidase activity increased between January and February and then remained stable until May. When monitored during the ripening process (January harvest), xylanase activity was constant, and xylosidase activity reached a peak at the climax of ethylene evolution and cellulase activity. Xylanase, which originated from Trichoderma viride and was added to the medium in which avocado discs were incubated, induced ethylene evolution.     

Adaptive traits of bark and ambrosia beetle-associated fungi

A phenotype is the expression of interactions between species genotype and environment. We quantified the contributions of ecological and phylogenetic associations to phenotypic variation in Geosmithia fungi. Geosmithia are symbiotic beetle-associated saprotrophs with a range of life histories and host specificities, including obligate nutritional beetle mutualists (ambrosia fungi) and phytopathogens. We hypothesized that: (1) species phenotypes are better explained by their ecology than by their phylogenetic relationships; (2) niche specialization was accompanied by enzymatic capability losses; and (3) ambrosia Geosmithia species have higher nutritional quality and antibiotic capabilities than species with facultative symbioses. Our results confirmed that long-term co-evolved specialists have reduced metabolic breadth in comparison to generalists. Phytopathogenic G. morbida produces unique enzyme suites with affinity to ligno-cellulose. Mycelia of ambrosia fungi contain large amounts of oleic fatty acid with nutritive and possibly allelopathic function. Overall, our results indicate that Geosmithia ecology have greater effect on species phenotype than their phylogenetic relationships.     

Symbiotic interactions of culturable microbes with the nickel hyperaccumulator Berkheya coddii and the herbivorous insect Chrysolina clathrata

Chrysolina clathrata is a specialized phytophagous beetle feeding exclusively on the herbaceous nickel hyperaccumulating plant Berkheya coddii. These organisms appear impervious to the toxic levels of nickel in their environment. In the current study we aimed to identify microorganisms that may have symbiotic relationships with these organisms. Culture techniques were used to isolate bacteria and fungi from plants and the faeces of beetles reared under laboratory conditions. The identity of isolates was determined using morphology and molecular techniques. Several genera of filamentous fungi (Alternaria, Aspergillus, Bipolaris, Cladosporium, Epicoccum, Fusarium, and Penicillium), yeasts (Cryptococcus, Meyerozyma, and Rhodotorula), as well as endophytic bacteria (Bacillus and Lysinibacillus) were isolated from the leaves of the plant. We also selectively isolated yeasts (Candida, Cryptococcus, Debaryomyces, Meyerozyma and Wickerhamomyces) from the beetle’s faeces. Subsequently we determined the minimum inhibitory Ni-concentration (MIC) of all isolates. The endophytic bacteria, filamentous fungi and the yeasts Candida intermedia, Cryptococcus flavescens and Meyerozyma guilliermondii, showed notable Ni resistance, while the beetle’s gut seems to select for Ni resistant yeasts.     

Peanut Pod Invasion by Aspergillus flavus in the Presence of Meloidogyne hapla

''Argentine'', ''Early Runner'' and ''Florigiant'' peanut cultivars were grown in methyl bromide treated soil in field microplots inoculated with: (i) Aspergillus flavus or (ii) A. flavus + Meloidogyne hapla. Nematode infection produced heavy root galling and light pod galling equally on all cultivars. A. flavus, A. niger, Cephalosporium spp., Colletotrichum sp., Curvularia spp., Fusarium spp., Penicillium spp. and Trichoderma viride were isolated from shells and kernels. A significantly greater incidence and density of A. flavus was obtained from kernels of plants inoculated with both organisms than from kernels of plants receiving only the fungus. Differences were not significant, however, for incidence and density of A. flavus in shells or for the total of all fungal propagules in shells and kernels. Shells of ''Early Runner'' contained significantly greater incidence and density of A. flavus than the other two cultivars; also, kernels of this cultivar contained more fungal propagules than kernels of ''Argentine.'' A significantly larger number of total fungi was isolated from kernels of ''Argentine'' than from ''Florigiant.'' Aflatoxins were found only in two shell samples and not in kernels.     

Molecular analysis reveals lowbush blueberry pest predation rates depend on ground beetle (Coleoptera: Carabidae) species and pest density

Molecular gut-content analysis allows determination of pest predation by field-collected predators. Ground beetles (Coleoptera: Carabidae) common in lowbush blueberries may consume blueberry spanworm, Itame argillacearia (Packard) (Lepidoptera: Geometridae), and blueberry flea beetle, Altica sylvia Malloch (Coleoptera: Chrysomelidae), providing pest suppression. Using newly developed pest specific primers, laboratory feeding trials showed that the median detection time (MDT) for blueberry spanworm in the largest beetle, Carabus nemoralis O.F. Müller, was 3.7 h, whereas Poecilus lucublandus (Say) and Pterostichus mutus (Say) had MDTs between 27.1 and 31.6 h for both pests. At a field-site with high pest abundances, the probability of detecting blueberry spanworm and blueberry flea beetle DNA was greater in P. lucublandus, 26 and 39 % respectively, than in P.mutus, 8 and 20 % respectively. Only 0 and 1 % of P. lucublandus and P. mutus, respectively, tested positive for blueberry spanworm DNA at a second site with low abundance. At the first site, the probability of detecting pest DNA in both ground beetle species was positively related to pest density. Higher pest DNA detection rates and captures of ground beetles corresponded to field areas where significant pest reductions occurred from late May to early June. Conservation of predatory carabid beetles could lead to valuable biological control in lowbush blueberries.     

First aeromycological study in an avocado agroecosystem in Mexico

Avocados (Persea americana Mill.) are economically and nutritionally valuable despite their susceptibility to several fungal diseases. This study was conducted in an agroecosystem of avocado trees in México. The air samples were collected every week by gravimetric methods at a height of 2 m from the ground level. Fungal colonies were isolated and transferred onto PDA and identified using morphological methods. During air sampling, different meteorological variables were measured. The symptoms of avocado diseases were determined by visual observations. To the best of our knowledge, this is the first study on aeromycological characterization in an avocado agroecosystem. Thirty-two airborne fungal genera were identified; Fusarium (97.2 %) and Colletotrichum (94.4 %) were the most common fungal pathogens present in the atmosphere of avocado. In addition, seven genera of important phytopathogenic fungi of other crops (Alternaria, Capnodium, Pestalotia, Stemphylium, Rhizopus, Curvularia, and Phyllachora) were isolated. The maximum concentration of total fungi was observed in June (358 CFU m^?3) and the minimum in September (83 CFU m^?3). The total fungal concentrations were significantly negatively correlated with the temperature. The symptoms of five diseases of avocado, viz. anthracnose, scab, spot, canker trunk, and vascular wilt, were identified in the area study. It was observed that the symptoms of all the above-mentioned diseases were present in June; and in the same month, the fungal concentrations were highest.     

Extreme ecological stoichiometry of a bark beetle–fungus mutualism

1. Ecological stoichiometry theory was applied to investigate how a consumer contends with an extreme elemental mismatch between its food and its body via symbiotic facilitation. 2. The beetle Dendroctonus brevicomis LeConte develops in bark, a substrate extremely low in nitrogen (N) and phosphorus (P). Its survival there depends on interactions with mutualist and antagonist fungi. 3. This study found that mutualists transfer N and P from sapwood and phloem into bark, where beetles feed, whereas the antagonist moves these elements only to phloem, resulting in starvation of the insect. However, even with mutualists, N and P concentrations remained low in bark, resulting in low N and extremely low P concentrations in the beetle. 4. The N:P ratios found in D. brevicomis larvae were the highest thus far reported for beetles and among the highest for insects and invertebrates. This suggests that the beetle has evolved additional, nutrient‐sparing adaptations.