Presence and viability of Ceratocystis lukuohia in ambrosia beetle frass from Rapid ʻŌhiʻa Death‐affected Metrosideros polymorpha trees on Hawaiʻi Island

Rapid ?ōhi?a Death (ROD) is a fungal disease of ?ōhi?a lehua (Myrtaceae: Metrosideros polymorpha) caused by Ceratocystis lukuohia and C. huliohia. ROD is the aetiological agent of widespread mortality of this important tree on Hawai?i Island, but its epidemiology remains unclear. We investigated the prevalence and viability of C. lukuohia in ambrosia beetle frass in ROD‐affected ?ōhi?a trees. A total of 200 frass traps were placed onto C. lukuohia‐infected ?ōhi?a at four locations on the east side of Hawai?i Island. Frass was collected and screened for the presence of C. lukuohia DNA using a diagnostic qPCR assay. In addition, frass samples were screened for viability by carrot baiting. All trapped beetles were of the genus Xyleborus, with the majority being the non‐native X. ferrugineus. Of the frass samples tested, 62% contained C. lukuohia DNA and 17% of carrot baits were positive for the fungus. These results indicate that ambrosia beetle frass releases C. lukuohia into the environment. We discuss the potential role infested frass could play in the ROD pathosystem.     

Screening of Metrosideros polymorpha (‘ōhi‘a) varieties for resistance to Ceratocystis lukuohia

Stands of the landscape‐dominant tree, Metrosideros polymorpha (‘ōhi‘a) on the Island of Hawai‘i, HI., USA, are dying from a phenomenon known as rapid ‘ōhi‘a death (ROD). Approximately 180,000 acres of forest have already been impacted by the disease, the majority of which is attributed to C. lukuohia, the more aggressive of the two Ceratocystis species responsible for ROD. Three isolates of C. lukuohia were compared for differences in aggressiveness. Mean disease severity and mean number of days to death did not differ among the three isolates of C. lukuohia. A single isolate was selected for use in a resistance screening of 128 plants across four varieties of M. polymorpha occurring on Hawai‘i Island: incana, glaberrima, polymorpha and newellii. Disease severity was lower in early‐successional var. incana (38.84%) and riparian var. newellii (36.11%) compared to late‐successional var. glaberrima (61.27%) and high‐elevation var. polymorpha (70.27%). Var. incana and var. newellii also had the lowest mortality (63% and 77%, respectively) while var. glaberrima (86%) and var. polymorpha (100%) had the highest mortality. Eighty‐five per cent of the plants that died did so within 49 days post‐inoculation and mean number of days to death was significantly higher for var. glaberrima (46) compared to var. polymorpha (31). The results of this preliminary study suggest that some form of resistance might be present in natural populations of at least one variety of M. polymorpha on Hawai‘i Island and should be investigated more intensively.     

First report of Ceratocystis platani in Albania

The fungus Ceratocystis platani was detected in various localities of the Gjirokastër prefecture in southern Albania, where it was causing widespread mortality on Platanus orientalis trees. The identification of the fungus was based on both morphological characteristics in culture and DNA sequencing. The pathogenicity of C. platani was confirmed in inoculations on seedlings of P. orientalis. To our knowledge, this is the first report of C. platani in Albania.     

Biscogniauxia nummularia infecting beech (Fagus sylvatica) trees and sympatric plants of the sedge Carex brevicollis

Biscogniauxia nummularia is known for its association with beech (Fagus sylvatica), on which it occurs as a saprotroph and a pathogen causing strip cankers following water stress. This fungus has also been reported as a dominant endophytic species in plants of the sedge Carex brevicollis growing in the understory of beech forests and adjacent grasslands in Sierra de Urbasa (Navarre, Spain). In this area, stromata of B. nummularia were observed in dead and living wood of beech trees at several locations where plants of C. brevicollis also contained the fungus as an endophyte. Pure cultures obtained from stromata of B. nummularia on F. sylvatica trees were compared to endophytic isolates from symptomless C. brevicollis. Culture morphology and micromorphology as well as rDNA sequences of the internal transcribed spacer (ITS) regions were identical, suggesting that B. nummularia from beech can also live endophytically in C. brevicollis. It is unknown whether the endophytic strains of Carex might have a role as an inoculum source for the infection of beech trees, or whether they represent a dead end in the life cycle of the fungus.     

Ambrosia beetles (Coleoptera: Curculionidae) can directly transmit the fungal pathogens responsible for Rapid ʻŌhiʻa Death

The ecologically and culturally vital tree species, ʻōhiʻa lehua (Metrosideros polymorpha), is threatened by the fungal pathogens Ceratocystis lukuohia and Ceratocystis huliohia, the causal agents of the disease complex called Rapid ʻŌhiʻa Death (ROD). Four invasive ambrosia beetle (Coleoptera: Curculionidae: Scolytinae) species in the Xyleborini tribe colonize ROD Ceratocystis-infested ‘ōhiʻa and produce inoculum through their frass; however, the potential for direct transmission of the ROD fungi by these beetles was unknown. We fulfilled Leach's rules to support insect transmission of ROD by documenting the visitation of these ambrosia beetles to healthy ‘ōhiʻa trees, culturing C. lukuohia and C. huliohia from the ROD-associated beetles using three different collection methods at multiple study sites, and challenging healthy ʻōhiʻa seedlings with beetles that were exposed to both C. lukuohia and C. huliohia cultures. We documented all four invasive ROD-associated ambrosia beetle species including Xyleborinus saxesenii, Xyleborus affinis, Xyleborus ferrugineus, and Xyleborus perforans to regularly visit healthy ʻōhiʻa trees on sticky traps. Viable Ceratocystis propagules were isolated from all species, and C. lukuohia was most commonly isolated of the two ROD-causing fungi. Consistently across all collection techniques, ROD Ceratocystis spp. were detected on just under 3% of all assayed beetles, with the highest detection rate from X. affinis. All four beetle species were capable of directly transmitting both pathogens to healthy ʻōhiʻa seedlings with a high rate of transfer. Ceratocystis spp. are highly virulent pathogens in trees, and a single inoculation can result in tree death, therefore any direct transmission is a cause for concern. After meeting the criteria of Leach's rules, we propose that Xi. saxesenii, X. affinis, X. ferrugineus, and X. perforans are vectors of C. lukuohia and C. huliohia, particularly in areas of high ROD pressure and tree stress.     

Occurrence of brown root rot in Ocean Expo Park,Okinawa, Japan: Infection route and preventive control measures

Ocean Expo Park, established in 1976 on subtropical Okinawa Island, Japan, has approximately 8000 planted or naturally grown trees in an area of 77.2 ha. Windfall of these trees occurs mainly due to typhoons; wood chips made from the trunks and roots of windfall trees are commonly used as mulching material throughout the park areas, despite possible infection by pathogenic fungi such as Phellinus noxius, the pathogen causing brown root rot. P. noxius is distributed in tropical and subtropical areas worldwide and has a broad host range. The fungus can infect host plants with mycelia via root-to-root contact or with basidiospores. Here, we report the current status of tree damage caused by brown root rot in the park and infer the infection routes of P. noxius based on microsatellite markers. Among a total of 294 trees surveyed in the park, P. noxius was isolated from 13 trees of five species. P. noxius was observed more frequently on Ficus microcarpa than on other tree species. Ficus superba var. japonica was identified as a new host species, and Bombax ceiba was first recorded as a host plant of P. noxius in Japan. Among the 13 P. noxius isolates from the diseased trees, 2 isolates from adjacent diseased trees were considered to be of the same genet, while the other 11 isolates were different genets, indicating that infection by basidiospores occurred frequently among trees in the entire park, although spread via root systems also occurred between at least two adjacent trees. An identical genet was not detected at distant locations, which suggests that the disease does not spread via chips. However, mulching with chips can lead to wood decay due to decaying fungi other than P. noxius. Therefore, in accordance with the precautionary principle, the park currently prohibits the chipping and mulching of windfall or damaged trees and is considering disposal methods such as burning.     

Tinctoporellus epimiltinus,a causal fungus of butt rot of Japanese cypress

One of the causal fungi of butt rot of Chamaecyparis obtusa in Kyushu Island, Japan, was identified as Tinctoporellus epimiltinus from cultural characters, and by mating and inoculation tests. The cultural characters of the isolates obtained from butt rot of C. obtusa and T. epimiltinus isolated from the basidiocarp tissue and from decayed wood were examined. The morphological characters and chemical reactions of the isolates from butt rot were typical for T. epimiltinus. Di‐mon mating tests were performed between five monospore isolates of T. epimiltinus and five heterokaryotic isolates from the butt rot. All of the monospore isolates examined were heterokaryotized by the isolates from butt rot. Tinctoporellus epimiltinus isolates were inoculated on roots of 24‐year‐old C. obtusa. Butt rot was observed on all inoculated trees 1–2 years after the inoculation, and the inoculated fungus was re‐isolated from the decayed parts of the inoculated trees.     

Field survey,isolation, identification and pathogenicity of Phytophthora species associated with a Mediterranean‐type tree species

Corymbia calophylla (marri), a keystone tree species in the global biodiversity hot spot of southwestern Australia, is suffering decline and mortality associated with a canker disease caused by the endemic fungus Quambalaria coyrecup. Phytophthora species are frequently isolated from the rhizosphere of C. calophylla, and a hypothesis is that Phytophthora root infection is predisposing C. calophylla to this endemic canker pathogen. Field surveys were conducted in both anthropogenically disturbed and undisturbed C. calophylla stands, from where a total of 100 rhizosphere soil samples, from both healthy and cankered trees, were collected. Phytophthora species were isolated from 26% of the samples collected, with Phytophthora incidence significantly higher on disturbed stands than in natural forests (73% and 27%, respectively). Five Phytophthora species were recovered, including P. cinnamomi, P. elongata, P. multivora, P. pseudocryptogea and P. versiformis. Under‐bark inoculations with the Phytophthora isolates caused significant lesion lengths in excised C. calophylla stems. Corymbia calophylla response to pot infestation trials in the glasshouse varied between Phytophthora species and isolates, with isolates of P. cinnamomi and P. multivora causing a significant reduction in seedling root volume and often leading to seedling death. This study demonstrates that root disease caused by Phytophthora species, especially P. cinnamomi and P. multivora, has the ability to adversely affect C. calophylla health. This study leads the way to do a dual inoculation trial with the canker pathogen Q. coyrecup, and different Phytophthora species to investigate if Phytophthora root infection predisposes C. calophylla to this canker disease.     

Root‐colonizing ophiostomatoid fungi associated with dying and dead young Scots pine in Poland

Ophiostomatoid fungi are carried by various bark beetles. However, very little is known about the role of these fungi in conifer roots. We studied ophiostomatoid fungi in roots of dying and dead Pinus sylvestris trees and tested the potential phytotoxicity of some isolates using a sensitive bioassay with Lepidium sativum in Poland. Fungi were identified based on their morphology and DNA sequencing. Three ophiostomatoid fungi, Leptographium procerum, Sporothrix inflata and Ophiostoma pallidulum, were isolated from the roots. The most abundant soil‐borne fungus, S. inflata, and relatively rare O. pallidulum were isolated for the first time from roots of dying and dead pine trees. The frequency of S. inflata and O. pallidulum correlated with tree decline. The fungi were isolated more frequently from roots of dead than dying trees. Sporothrix inflata and O. pallidulum slightly reduced the stem and root growth of L. sativum. Leptographium procerum reduced more significantly root than stem growth. This species reduced root elongation 32–54% after 10–17 days of incubation.     

Platypus cylindrus,a vector of Ceratocystis platani in Platanus orientalis stands in Greece

The ambrosia beetle Platypus cylindrus was very common in stands of Platanus orientalis trees in Greece, infesting trees already infected by Ceratocystis platani. The fungus C. platani was isolated from 59% of adult beetles of P. cylindrus collected on the wing in a heavily infected stand of P. orientalis. Transmission of C. platani was demonstrated in 80% of P. orientalis seedlings challenged with P. cylindrus beetles, which were previously fed in cultures of the fungus. In most of the cases, the beetles had bored tunnels in stems, where abundant perithecia of C. platani were observed. Platypus cylindrus adults regularly visited artificially wounded P. orientalis trees in a natural stand. Although this ambrosia beetle normally infests stressed or dead trees, it appears to play a role as a vector of C. platani, especially by visiting wounded trees.     

Occurrence of Phytophthora cinnamomi in cork oak forests in Italy

An increasing decline and mortality of cork oak trees have been recently observed in central Italy and Sardinia Island. Following surveys conducted in three declining cork oak forests, a Phytophthora species was consistently isolated from soil samples collected from trees displaying different level of decline. Based on morphological features, growth rates at different temperatures and analysis of DNA sequences of the ITS region, all isolates were identified as Phytophthora cinnamomi Rands. This pathogen caused large brownish lesions on inoculated freshly cut branches of cork oak. It was re‐isolated from all infected tissues. These findings represent the first report of P. cinnamomi on cork oak trees in Italy.     

Natural infection of Fraxinus excelsior seeds by Chalara fraxinea

The occurrence of Chalara fraxinea, the fungus responsible for dieback of European ash (Fraxinus excelsior), was investigated in the current and previous seed years collected from symptomatic trees in Latvia and Sweden using molecular techniques (DNA extraction, ITS‐PCR, Sanger sequencing). Molecular analysis of seeds revealed the presence of 30 different fungal taxa. Chalara fraxinea was detected in 8.3% of seeds tested from the current year originating from Latvia. The presence of C. fraxinea in seeds of F. excelsior is of great concern to phytosanitary protection authorities in countries outside the current zone of infestation.     

Mycobiota in needles of Abies alba with and without symptoms of Herpotrichia needle browning

The ascomycete Nematostoma parasiticum (syn.: Herpotrichia parasitica) is commonly perceived as the causal agent of the so‐called Herpotrichia needle browning in silver fir (Abies alba). However, its fruitbodies are rarely present on symptomatic needles, which are also colonized by many presumably saprotrophic fungi. We compared the internal colonization of healthy and symptomatic needles on two sites in Poland. In addition, the endophytic mycobiota in needles of various age was recorded on two other sites without disease symptoms. Fungi were isolated from 95.6% of the dead needles and from 62.9% of the living needles on symptomatic trees, whereas on healthy trees, only 45.0% of the needles were colonized internally. Colonization frequency increased with needle age. From a total of 2017 isolates, 116 fungal taxa were identified. Frequency of many species was influenced by needle type. Anthostomella formosa, Gloeosporidiella sp., Hypoxylon fragiforme, Xylaria hypoxylon and X. polymorpha were the most common fungi isolated from living needles. In symptomless living needles, fungi occurred significantly more often in the basal than in the apical parts. In dead needles, the most common fungi were Alternaria alternata, Paraconiothyrium sporulosum, Fusarium sp., Mollisia cinerea, Rhizoctonia sp., Rhizosphaera oudemansii, Thysanophora penicillioides, Xylaria hypoxylon and X. polymorpha. Rhizoctonia sp. was the most frequently isolated fungus in dead needles (23.4%) but occurred rarely also in living needles (0.3–1.1%). The supposed pathogen N. parasiticum was detected only sporadically (at most in 0.6% of the needles). Our findings demonstrate the need for understanding the role of Rhizoctonia sp. in Herpotrichia needle browning disease aetiology.     

Characterization of host–fungus interactions among wood decay fungi associated with Khaya senegalensis (Desr.) A. Juss (Meliaceae) in Singapore

Tree pruning creates wounds that are amenable for wood decay fungi colonization. To characterize the dynamic host–fungus interactions at this location in Senegal mahogany (Khaya senegalensis), in vitro and in vivo pathogenicity tests were conducted with wood decay fungi associated with this tropical tree species. Fomitiporella caryophylii, Hymenochaete murina and Phellinus noxius isolates were included in this experiment following their frequent isolation from Senegal mahogany pruning wounds. The evaluated isolates demonstrated unique host interactions in laboratory tests that suggest equally divergent prognoses for living Senegal mahoganies affected by these fungi. Although all evaluated fungal isolates successfully breached naturally induced reaction zones, P. noxius alone caused significant mass loss to incubated wood blocks. In addition, P. noxius caused extensive wood decay after inoculation in living hosts, successfully illustrating Koch's postulates for this host–fungus relationship. The wood decay ability, invasiveness and facultative parasitism demonstrated by P. noxius suggest its dominant role in wood decay columns below pruning wounds on living Senegal mahoganies. These results highlight the importance of characterizing specific host–fungus interactions and their implications for wood decay severity below pruning wounds in living trees.     

Diversity of Cryphonectria parasitica in callused chestnut blight cankers on European and American chestnut

Infection of American and European chestnuts with the chestnut blight fungus Cryphonectria parasitica results in the formation of cankers, lesions caused by the growth of mycelia within bark tissue of the host plant. Infection of the fungus with Cryphonectria hypovirus 1 (CHV‐1) results in conversion of the mycelial phenotype from virulent to hypovirulent, thus allowing production of callus around cankers as a reaction by infected trees, rendering active into inactive cankers. In this study, we sampled one USA and six European chestnut stands and assessed frequency of hypovirulent C. parasitica and diversity of vegetative compatibility (vc) types present in calluses and randomly sampled cankers. Callused cankers on C. dentata at West Salem in the USA yielded significantly more hypovirulent C. parasitica isolates compared with four sampled populations on C. sativa, while all six sampled European populations did not show any statistically significant differences among themselves. We observed no correlation between hypovirulence frequencies in randomly sampled cankers and calluses, as well as no correlation of C. parasitica vc type diversity in calluses and residential populations of the fungus. Furthermore, even though we have observed calluses with more than one vc type, they do not occur regularly. Even when present in C. parasitica populations with high vc type diversity, no more than three different vc types were observed in a single callus.     

First report of Cryptosphaeria pullmanensis as causal agent of Cryptosphaeria canker of Populus nigra in Iran

A diatrypaceous fungus was isolated consistently from cankers on Populus nigra trees showing dieback symptoms in Kohgiluyeh Boyer‐Ahmad, Zanjan and Esfahan provinces in Iran. Morphological characteristics and phylogenetic analyses of the ITS region of the rDNA identified the taxon as Cryptosphaeria pullmanensis. Pathogenicity tests conducted in potted 3‐month‐old cuttings of P. nigra confirmed Koch's postulates and revealed that C. pullmanensis caused canker on this host. This is the first report of C. pullmanensis causing a canker disease on P. nigra in Iran.     

Leptoglossus occidentalis and Diplodia pinea: a new insect‐fungus association in Mediterranean forests

Leptoglossus occidentalis, an insect native to North America, was inadvertently introduced into Italy about 1999. The insect damages the cones of conifer trees, especially Pinus pinea (Italian Stone pine). Pinus pinea is also affected by Diplodia pinea, a fungus native to Italy, which is becoming an increasing threat because pine trees are becoming more susceptible to it as a consequence of global warming. Because the insect and the fungus both have the pine cones as a common habitat, a possible interaction between them has been postulated. The aim of this study was to ascertain whether L. occidentalis and D. pinea interact on P. pinea cones. The interaction was studied using real‐time PCR on a group of naturally infected insects collected from a forest, and a group raised in the laboratory and artificially inoculated with D. pinea conidia. Molecular analysis showed that D. pinea DNA occurred on both naturally infected and inoculated insects, but with significant differences between the two groups. The rapid and sensitive molecular technique made it possible to detect D. pinea DNA on the bodies of the insects, and to show that the native D. pinea occurred on the exotic insect.     

Long‐term field study of transgenic hypovirulent strains of Cryphonectria parasitica in a forest setting

Transgenic hypovirulent strains of Cryphonectria parasitica, the chestnut blight fungus, engineered to contain a chromosomally integrated full‐length infectious cDNA copy of virulence‐attenuating hypoviruses, differ from natural hypovirulent strains in the ability to transmit hypoviruses to ascospore progeny and with 100% efficiency through asexual spores. We report the results of a long‐term field study that examined whether these properties result in enhanced hypovirulence establishment, dissemination and persistence under field conditions. Informed by previous field results using a severe hypovirus, this study that employed 144 American chestnut trees was designed to provide improved inoculum formulation and delivery and to include the use of a mild hypovirus isolate (less debilitating) CHV‐1/Euro7 in an attempt to increase dissemination. Isogenic transgenic hypovirulent (TG), non‐transgenic cytoplasmic hypovirulent (CH) or virus‐free virulent (V) treatment strains were applied to artificially initiated and natural C. parasitica cankers three times each year for 7 years. Reservoirs of treatment inoculum also were initiated and refreshed annually for the first 6 years of the study. Sampling of 111,000 individual ascospores from 4,500 perithecia confirmed hypovirus‐containing spermatia successfully transmitted TG hypoviruses to ascospore progeny under field conditions. Surprisingly, TG ascospore progeny were recovered 3 years after the last annual application of treatment inoculum. Repeated sampling of over 440 cankers revealed dissemination of both CH and TG hypovirulent strains. However, no significance differences in establishment or dissemination were observed for the two hypovirulent strains. The results are discussed in terms of the contribution of ascospore progeny to infection, competition by endemic virulent C. parasitica, size of inoculated trees and the biological control potential of TG hypovirulent strains.     

Local epidemiology of the wood decay agent Laetiporus sulphureus in carob stands in Sicily

The basidiomycete Laetiporus sulphureus (Bull) Murrill is a forest pathogen causing brown cubical heart rot in a broad range of host trees. Despite its wide distribution and importance, studies aimed at understanding the epidemiology of the fungus in specific areas or hosts are lacking. In this study, an incidence of L. sulphureus as high as 34% was determined through molecular analysis of wood samples collected from 70 carob (Ceratonia siliqua L.) trees in the South‐west of Sicily, Italy. A phylogenetic analysis of Internal Transcribed Spacer (ITS) sequences indicated that all carob isolates belonged to the cluster E of Laetiporus taxonomy. Ten molecular markers based on Single Sequence Repeats (SSRs) designed on the L. sulphureus genome were developed, and isolates were genotyped through High‐resolution Melting (HRM) analysis. High gene diversity (0.581), no correlation between fungal genotype and host tree species and significant correlation between spatial and genetic distance were observed, suggesting an important role of basidiospores in the epidemiology of the fungus and a risk of transmission from a host tree species to the others. Finally, from a prognostic perspective, significant differences among isolates in terms of mycelial growth suggest that in addition to an accurate identification, a phenotypic characterization of isolates affecting trees may also be important.     

A preliminary report on decay and canker of Acacia richii caused by Inonotus rickii in China

An epidemic wood‐decaying fungus, Inonotus rickii, originating from South America, has spread to the Mediterranean area and southern China. We report the first observation of this fungus on Acacia richii in Panzhihua, southern China. It causes serious canker and decay on urban trees in the investigated area. In addition, phylogenetic analysis showed that the fungus from Panzhihua is closely related to isolates from Europe, South America, Mianyang and Hainan of China. Moreover, it was concluded that the isolates of the species previously reported from the USA do not belong to I. rickii.