Investigating bark, ambrosia and nitidulid beetle (Coleoptera: Scolytinae and Nitidulidae) communities and their potential role in the movement of Ceratocystis manginecans in commercial forestry plantations in Riau, Indonesia

Inoculations of two Acacia populations were used to estimate the level of genetic control for tolerance to Ceratocystis canker and wilt disease following inoculation with the causal pathogen, Ceratocystis manginecans. The first screening utilised a range of clones commonly established in Vietnam along with new candidate clones and provided evidence that variation in wilt symptoms is under a moderate level of genetic control in current deployment populations. Subsequent assessments of tolerance in a pedigreed Acacia auriculiformis progeny trial indicated that internal lesion length assessments were less influenced by seedling height and produced higher heritability estimates than external lesion length assessments. Heritability estimates for damage to detached phyllodes following inoculation increased from 2 to 4 weeks, at which point many leaves were entirely damaged. Assessments of phyllode damage were more heritable than assessments of stem damage, and positive genetic correlations between stems and phyllode damage indicate that families will rank similarly for both traits. If validation trials confirm the results of this study in larger trees, screening as described herein may be used to develop acacia breeds with greater disease tolerance.

Species of Ceratocystis are well-known wound related pathogens of many tree species, including commercially planted Acacia spp. Recently, several Ceratocystis isolates were collected from wilting A. mangium in plantations in Indonesia. The aim of this study was to identify these Ceratocystis isolates and to investigate their ability to cause disease on two plantation-grown Acacia spp. using greenhouse and field inoculation experiments. For identification, morphological characteristics and comparisons of DNA sequence data for the ITS, β-tubulin and TEF 1-α gene regions, was used. Ceratocystis isolates were identified as C. manginecans, a serious pathogen of mango trees in Oman and Pakistan and a previously undescribed species, described here as C. acaciivora sp. nov. Both fungi produced significant lesions in inoculation experiments on A. mangium and A. crassicarpa, however, C. acaciivora was most pathogenic suggesting that this fungus is the primary cause of the death of trees under natural conditions.

Many fungi live in close association with insects, and some are specifically vectored by them. One of the best examples is found in the so-called Ophiostomatoid fungi, including species of Ceratocystis and other genera in the Ceratocystidaceae. Our understanding of vectorship in these fungi is based predominantly on either their frequency of isolation from insects or the success with which these fungi are isolated from their insect vectors. The fact that Ceratocystis species mostly have casual as opposed to highly specific relationships with their insect vectors makes it difficult to prove insect vector relations. In order to provide unambiguous support for Ceratocystis species being vectored by insects, we interrogated whether genotypes of the tree pathogen, Ceratocystis albifundus, would be shared between isolates retrieved from either infected trees or nitidulid beetles. Ceratocystis albifundus isolates were collected from nitidulid beetles and from naturally occurring wounds on trees in the Kruger National Park (KNP) in South Africa. The genotypes of these isolates were then determined using eight microsatellite markers, and they were compared using a haploid network analyses. A high frequency of Multi-Locus Haplotypes (MLHs) derived from nitidulid beetles was found to be shared with those from wounded trees across the KNP. This provides robust support showing that nitidulid beetles play an important role in the dispersal of C. albifundus in the KNP.

Many species of sap beetles have been implicated as vectors of the oak wilt pathogen, (Ceratocystis fagacearum), but the species responsible for most aboveground transmission of the fungus is unknown. The abundance of adult sap beetle species inhabiting <7-day-old, artificially made wounds on healthy oaks during Apr.–June of 2 years in Minnesota, and the frequencies with which viable propagules of the fungi were present on each, were determined. Sap beetles were most abundant on wounds created in May, and 1–3-day-old wounds yielded higher numbers of insects than 4–6-day-old wounds. Of the beetles collected, >95% were Colopterus truncatus and Carpophilus sayi. C. fagacearum was isolated from 75% of 594 adults assayed for the fungus. The average number of viable propagules for pooled data of individuals of each species ranged from <100 to 18,000. Frequencies of fungus isolation differed by days after wounding, study location, and sap beetle species. These results, when coupled with previous findings on predominant species associated with oak wilt fungi mats, support the hypothesis that Co. truncatus and Ca. sayi are the principal sap beetle species transmitting C. fagacearum from diseased to healthy oaks in Minnesota. FOR. SCI. 50(6):757–764.

Rapid ʻŌhiʻa Death (ROD) is a fungal disease of ʻōhiʻa lehua (Myrtaceae: Metrosideros polymorpha) caused by Ceratocystis lukuohia and C. huliohia. ROD is the aetiological agent of widespread mortality of this important tree on Hawaiʻi Island, but its epidemiology remains unclear. We investigated the prevalence and viability of C. lukuohia in ambrosia beetle frass in ROD‐affected ʻōhiʻa trees. A total of 200 frass traps were placed onto C. lukuohia‐infected ʻōhiʻa at four locations on the east side of Hawaiʻi 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.

Classification of pine wilt disease at different infection stages by diagnostic hyperspectral bands

A possible centre of diversity in South East Asia for the tree pathogen, Ceratocystis manginecans

Oak wilt is slowly expanding in the northeastern United States. Several nitidulid beetle species are known vectors of the fungus [Bretziella fagacearum (Bretz) Z. W. De Beer, Marinc., T. A. Duong, and M. J. Wingf (Microascales: Ceratocystidaceae)] that causes this disease, acquiring spores from fungal mats on infected trees and transmitting them to uninfected trees. Survey and fungal isolation from captured nitidulid beetles could be an important tool for detecting the presence of this disease in a geographic area not previously known to have oak wilt. In preparation for monitoring activities in such areas, two trapping studies were conducted in the northeastern United States: 1) trap test comparing the efficacy of wind-oriented pipe, multiple-funnel, and modified pitfall traps for nitidulids and 2) wet and dry collection cup comparison. Lures were a combination of nitidulid pheromones and fermenting liquid. Results support the use of multiple-funnel traps over the other two trap types, for both targeted species-specific surveys and community sampling. More total nitidulids, Colopterus truncatus (Randall), and Glischrochilus fasciatus (Olivier) were captured in wet collection cups compared with dry cups. Twenty-seven fungal species were isolated, none of which were B. fagacearum. Many fungi isolated from beetles were plant pathogens, indicating that in addition to the oak wilt fungus, sap beetles may contribute to the spread of other plant diseases.

Ceratocystis wilt and canker disease has devastated Acacia mangium plantations in south-east Asia. Current screening methodologies to identify resistant or tolerant germplasm use potted plants in a greenhouse as a preliminary screening to select material for field trials, but these tests are time-consuming, motivating a search for rapid screening protocols. In this study, inoculation procedures were tested on three species of Acacia, viz. A. mangium, A. crassicarpa and a hybrid of A. auriculiformis and A. mangium, using three isolates of Ceratocystis manginecans. Mycelial plugs were compared with spore suspensions as inoculum to infect artificial wounds on the stems of A. mangium potted plants. The rapid screening protocols involved inoculation of stem segments with mycelial plugs and detached phyllodes with a spore suspension, with susceptibility measured by lesion length on the stems or necrosis length on the phyllodes. Both mycelial plugs and spore suspensions produced a similar level of disease incidence, so either inoculum form can be used for an assay. The stem segments were prone to contamination by other fungi and to desiccation, while results from the potted plant and phyllode protocols showed similar trends of susceptibility among the Acacia clones and species. The ease, rapidity, and reproducibility of the phyllode inoculation protocol makes it a potential replacement for inoculation of potted plants as a preliminary screening protocol to identify disease tolerant A. mangium germplasm prior to field screening.

Summary The nematode associations of two nitidulid beetles, Epuraea ocularis and Lasiodactylus pictus, and their habitat (substrate) were examined in an experimental stand in Kyoto, Japan. Beetles were collected in October 2020 using banana traps. Molecular and morphological identification recognised two nematode species: Sheraphelenchus sucus and Chylorhabditis epuraeae. Sheraphelenchus sucus was isolated exclusively from E. ocularis, while C. epuraeae was isolated from both beetle species. Dauer juveniles (DJ) of S. sucus were isolated from the substrate, rotten banana. The typological characters of S. sucus DJ are illustrated and photo-documented, and S. heterophallus n. comb. (= Aphelenchoides heterophallus Steiner, 1934 and Parasitaphelenchus heterophallus (Steiner, 1934) Rühm, 1956), recognised during a literature survey, is proposed.

Earwigs (Dermaptera), such as Forficula auricularia L., are important euryphagous predators for a wide variety of prey and can markedly influence the populations of orchard pests. Most previous studies on earwig feeding behaviour have not used adult beetles of the prey species; few researchers have focused on prey preference in earwigs. Some fragments of beetle exoskeleton and an earwig adult, Anisolabella marginalis (Dohrn), were found in the same cage, where adults of ambrosia beetle, Euwallacea interjectus (Blandford), were emerging from the logs of a fig tree infected with Ceratocystis canker (fig wilt disease). Thus, A. marginalis was suspected of being a predator of E. interjectus. To shed light on this issue, in the laboratory, we set up a test arena and observed and recorded behavioural interactions between A. marginalis and E. interjectus. E. interjectus was collected from the logs of fig trees and reared on an artificial diet, along with six different ambrosia beetle species, which were collected from a trap (baited with ethanol) and a fallen maple tree. A series of laboratory experiments demonstrated that A. marginalis is actually a predator of E. interjectus and other species of ambrosia beetle, indicating its a potential for use in effective pest control in the field. The predators frequently consume and tend to select their prey depending on prey size, rather than sex and beetle species. Furthermore, earwigs have alternative predatory strategies for dealing with seven different species, although they use their forceps to cut the body of most tested beetles.

Dalbergia tonkinensis is a rare and valuable rosewood which is being domesticated in home gardens in north Vietnam. Concern for the viability of this new industry has arisen with growers reporting the recent death of trees. This study identified a new wilt disease causing large bole cankers, associated blue staining to the wood, wilted canopies and tree death in home gardens and plantations. From βT1a and βT1b sequence analysis, the Ceratocystis isolates causing wilt disease in D. tonkinensis belong to Ceratocystis manginecans and were shown to be pathogenic with D. tonkinensis seedlings in a nursery trial. They were also able to form stem cankers in Acacia and Eucalyptus. Cankers in 2–11 year-old trees were associated with wounding, primarily from pruning of branches to improve tree form and drilling of trunks in older trees to assess heartwood formation. To mitigate against further damage, it is recommended that pruning be restricted to the dry season and that the practice of drilling trunks be kept to a minimum.

Wilt disease has become a threat to Acacia plantations in SE Asia. There are about 1.3 million ha of Acacia auriculiformis, A. mangium and Acacia hybrid plantations in Vietnam that require management of wilt disease caused by Ceratocystis manginecans. To identify options for management, we examined affect of pruning (tree age, pruning time and pruning technique) and site traits (altitude, slope and position) on disease expression from natural infections. Ceratocystis wilt disease and disease indices were more serious in younger than older pruned trees. Pruning in the rainy season resulted in heavier infection by Ceratocystis than pruning in the dry season. Decreasing bark tearing during pruning reduced the levels of infection and disease. Tip pruning of branches reduced disease compared to pruning close to the trunk. The interaction effects of terrain factors showed that A. auriculiformis and Acacia hybrid plantations were most severely affected when trees were planted in foot-hills, at altitudes below 300 m and on slopes below 15o. Results for the pruning time and pruning technique trials have application for reducing the impact of Ceratocystis wilt disease in Acacia plantations in Vietnam.

High-resolution melting curve analysis: A detection assay for Ceratocystis eucalypticola and C. manginecans in infected Eucalyptus

Psidium guajava is a widely grown fruit tree of Asia for food and medicinal purposes. Also being reported to have anti-inflammatory, antimicrobial, antioxidant, antidiarrheal, antimutagenic properties (Somu, 2012). In April 2018, quick decline disease of guava was observed in orchards of Sheikhupura, Lahore, Faisalabad, Kasur and Chiniot districts of Punjab, Pakistan. Approximately 68% of the trees were found declined with mummified fruits. Initial infection symptoms appeared as wilting of leaves, bark discoloration, followed by the leaf drooping, crown area discoloration, bark splitting, mummified fruits, dying of branches and lately whole tree death in weeks to months. The fungus formed a dark brown to black discoloration (3 to 5 cm wide and 7 to 9 cm long) in vascular bundles of P. guajava tree. Sixty-five samples of discolored wood from the main stem were collected, and pathogen was isolated using carrot bait method (Moller and DeVay, 1968). Isolation and purification were done on 2% Malt extract agar (MEA) plates incubated at 25 ± 2 °C in 12 h light and dark period. After 6 days of incubation, fungal hyphae, fruiting structures, sexual & asexual spores were observed on MEA plates. Black globose to subglobose ascomata with bases (151-) 200 (-278) µm in diameter with long neck (511-) 535 to 600 (-671) µm long, (23-) 28 to 39 (-47) µm wide at base, (13-) 13- 19 (-25) µm wide at tip and light brown to hyaline divergent ostiolar hyphae (50µm) were developed and produces hat-shaped hyaline ascospores 3 to 5 µm long and 6-7 µm (with sheath) and 4 µm (without sheath) wide. After 7 days, initially white mycelium turned into olivaceous green and produced primary phialidic conidiophore with emerging primary cylindrical hyaline conidia (7 to 12 × 4 to 6 µm), secondary conidiophore with emerging chain of secondary barrel-shaped hyaline conidia (9-) 10 to 12 (-13) µm long × (5-) 5 to 9 (-11) µm wide and dark brown dematiaceous chlamydospores conidia (12 ×10 µm) were observed. All morphological characteristics were consistent to the description of Ceratocystis manginecans (Van Wyk, et al., 2007). For further confirmation, from a purified isolate GWD10, genomic DNA was extracted. The internal transcribed spacer (ITS) and translation elongation factor 1-alpha (TEF 1-α) region were amplified with primer pairs ITS1/ITS4 and EF1/EF2 (Jacobs et al., 2004; White et al., 1990) respectively. Generated sequences (Accession Nos. MN 365128 & MT952139) on BLAST analysis showed 100% homology for ITS and TEF with Ceratocystis manginecans (Accession No., KC261852 CMW 13582 Voucher, NR-119532.1 type material, MH863135; EF433317, respectively) reported from Oman and Pakistan (Van Wyk et al., 2007 & Vu et al., 2019). For pathogenicity test, one-year-old healthy P. guajava plants were inoculated by making a T-shaped slit of 5 × 7.5 mm in the bark. Two weeks old cultures of GWD10, 5-mm mycelial discs were aseptically transferred and covered with moistened sterilized cotton swab followed parafilm to maintain humidity. Fifteen plants were inoculated with fungal cultures and five plants were inoculated with MEA plugs as controls. All plants were maintained at 25 ± 2 °C with 80 ± 5% relative humidity (RH) in greenhouse Initial bark discoloration developed after 14 days of inoculation. After 40 days of inoculation plants started wilting and dying, similar to the symptoms were observed in naturally infected trees. Control plants remained asymptomatic. To fulfill Koch's pustulates, the same pathogen was re-isolated from the test plants and identified on morphological features to GWD10. The pathogen has been associated with mango decline in Oman and Pakistan (Van Wyk et al., 2007), acacia wilt in Indonesia (Harrington et al., 2015) and siris wilt in Pakistan (Razzaq et al., 2020). P guajava is an important fruit and medicinal plant, and the infection of C. manginecans is a great concern to the producers of P. guajava (Harrington et al., 2015; Huang et al., 2003). To our knowledge, this is the first report of Ceratocystis manginecans causing quick decline of P. guajava worldwide.