Abstract Ceratocystis Canker and Wilt Disease (CCWD), caused by the fungal pathogen Ceratocystis manginecans, poses a significant threat to Acacia and Eucalyptus plantations in Indonesia. Infections typically occur through wounds on the main stems of trees, and infected trees, particularly Acacia, are often co‐infested by various wood‐associated beetles. The aim of this study was to identify the beetle species associated with Ceratocystis‐infected trees and to assess their potential role in dispersing C. manginecans. Beetle and frass samples were collected from Ceratocystis‐infected compartments of Acacia and Eucalyptus propagated as monocultures in Riau, Indonesia, either by using traps or by direct collection from infected trees. The samples were screened for the presence and viability of Ceratocystis propagules using a quantitative polymerase chain reaction‐high‐resolution melting analysis (qPCR‐HRMA) diagnostic tool and a carrot baiting technique, respectively. Fourteen beetle species, predominantly collected from traps, representing nine genera of the Scolytinae and Nitidulidae, were identified from the two plantation types. The qPCR tool detected the presence of Ceratocystis DNA on 13 of the 14 beetle species at concentrations as low as 0.015–0.0019 ng. However, viable Ceratocystis propagules could not be recovered from these beetles using culture‐based methods. Of the 105 frass samples screened, qPCR detected Ceratocystis DNA in 67 samples; however, only one Ceratocystis isolate was obtained using the carrot baiting method. The results of this study suggest that the beetles and beetle frass can harbour Ceratocystis propagules, as indicated by the detection of fungal DNA. However, the viability of the propagules and the ability of the beetles and/or beetle frass to spread C. manginecans requires further investigation.

In Indonesia and Malaysia, Eucalyptus pellita has replaced large areas of Acacia mangium plantations as the latter species is highly susceptible to C. manginecans. This strategy may not be effective in the long term as E. pellita is also susceptible to Ceratocystis wilt and canker disease though it has higher tolerance than A. mangium. Furthermore, the pathogen has the capacity to evolve and adapt to new hosts. To highlight the need for careful sanitation in nurseries and the potential impact of wounding during silvicultural operations, two experiments were conducted to (i) demonstrate the ability of cryptic Ceratocystis infections in nursery plants to develop into Ceratocystis wilt and canker disease after planting out and (ii) assess the risk of Ceratocystis infection and disease development from different wound types. In the nursery, three-month-old mini-cuttings of seven E. pellita clones were artificially wounded and inoculated with two isolates of Ceratocystis manginecans near the base of the stem. The disease incidence and lesion length were measured one month later, just prior to planting out. After four months of growth in the plantation, the trees were harvested and stems sliced longitudinally to measure the length of xylem discolouration. Six of 36 ramets of susceptible clones died and the length of xylem discolouration was significantly greater in susceptible clones than in tolerant clones. The second experiment was based on inoculation of 12-month-old plantation-grown trees of one clone of E. pellita with one isolate of C. manginecans using six different wounding methods. The inoculated wounds on the trees all produced xylem discolouration, except for those that only penetrated the outer bark. Disease incidence was greater at stem heights of 30 to 90 cm than on the basal stem or branch stub. The experiment emphasised the importance of minimising the risk of C. manginecans infection following wounding in the nursery and in the field as the discolouration is an indication of xylem blockage that can lead to tree mortality.

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

Abstract. Yunus NM, Maid M, Yong WTL, Anthony FE, Sudin M, Taylor PWJ. 2024. Identification and pathogenicity of Ceratocystis manginecans causing wilt disease on Acacia mangium in Sabah, Malaysia. Biodiversitas 25: 2170-2182. An alarming incidence of wilt disease has been reported in an Acacia mangium plantation in Ulu Kukut, Kota Belud District, Sabah, Malaysia. Infected trees exhibited symptoms such as severe wilting, sapwood discoloration or black lesion, and a fruity-sweet odor emanating from the fermentation exudate at the wound lesion. This is the first investigation of the causal wilt pathogen in a commercial A. mangium plantation in Ulu Kukut, located in the western region of Sabah. This study aimed to identify the causal fungal pathogen from infected A. mangium trees using morphological characterization and DNA sequence comparisons for the regions of Internal Transcribed Spacer (ITS), beta-tubulin 1 (bt1), transcription elongation factor-1 alpha (tef1), guanine nucleotide-binding protein subunit beta-like protein (ms204), and second largest subunits of RNA polymerase II (rpb2). The fungal isolates shared morphological characteristics with the wilt pathogen Ceratocystis sp., including a globose base with a long neck-ended tip with ostiolar hyphae. Sequence-based phylogenetic analysis confirmed the identity of Ceratocystis manginecans, distinguishing them from all other Ceratocystis species. Bioassays inoculating phyllodes and twigs of 1-year-old A. mangium trees confirmed that C. manginecans was the cause of wilt disease. Confirming the identity of the causal agent of the increasingly destructive and severe wilt disease aids in developing effective disease management strategies for Acacia-based plantations.

Ceratocystis manginecans has caused significant losses in forestry productivity in Indonesia and neighboring nations. It also infects horticultural trees, but the host range of individual isolates of C. manginecans is poorly studied. So, this study aimed to better understand the potential host range and evaluate aggressiveness against forestry and fruit tree species of C. manginecans isolated from various tree species in Indonesia. Five C. manginecans isolates, four from different tree species and one from the shot-hole borer Euwallacea perbrevis, were used to inoculate seven fruit and six forest tree species, including E. pellita and Acacia mangium. Many of the inoculated trees produced typical canker disease symptoms, such as rough, swollen, and cracked lesions on the bark, but some trees did not have any external symptoms. Mortality in the most susceptible clone of A. mangium was 40% within 8 weeks. Forest tree species were more susceptible than fruit trees, with the length of xylem discoloration ranging from 0.4 to 101 cm. In fruit trees, the average extent of xylem discoloration was lower, ranging from 0.4 to 20.5 cm; however, mortalities were recorded in two fruit tree species, Citrus microcarpa and Durio zibethinus. Host-isolate interaction was evident; isolate Ep106C from Eucalyptus pellita caused the greatest xylem discoloration in Citrus sp., whereas Hy163C from Hymenaea courbaril was the most damaging in D. zibethinus, Artocarpus heterophyllus, and Mangifera indica. Increasingly globalized food and fiber systems increase risk of disease spread, and the serious threat of C. manginecans incursions into countries where it is not present must be evaluated more thoroughly.

ABSTRACT The genus Ceratocystis contains a number of emerging plant pathogens, mostly members of the Latin American Clade (LAC), in which there are several unresolved taxonomic controversies. Among the most important are Brazilian pathogens in the C. fimbriata complex, C. manginecans and C. eucalypticola. Representatives of C. manginecans and C. eucalypticola from India and China, respectively, were shown to be fully interfertile in laboratory matings, and hybrids between the putative species were identified on Punica in India. An Indian tester strain was sexually compatible with representatives of what has been considered C. fimbriata on numerous hosts across Brazil. In this revision of the LAC, the name C. fimbriata is restricted to the widely dispersed Ipomoea strain, and C. manginecans is recognized as a Brazilian species that is important on Mangifera, Eucalyptus, and many other crops. C. mangivora and C. mangicola are also considered synonyms of C. manginecans. Based on phylogenetics and mating studies, two other Brazilian species are recognized: C. atlantica, sp. nov., and C. alfenasii, sp. nov., each with wide host ranges. Three new Caribbean species are recognized based on phylogenetics and earlier inoculation studies: C. costaricensis, sp. nov., on Coffea, C. cubensis, sp. nov., on Spathodea, and C. xanthosomatis, sp. nov., on the vegetatively propagated aroids Xanthosoma and Syngonium. Some of the other Ceratocystis species were based primarily on unique internal transcribed spacer (ITS) rDNA sequences, but the unreliability of rDNA sequences was demonstrated when intraspecific crossing of isolates with differing ITS sequences generated single-ascospore progeny with intragenomic variation in ITS sequences and others with new ITS sequences. Species recognition in Ceratocystis should use phenotype, including intersterility tests, to help identify which lineages are species. Although some species remain under-studied, we recognize 16 species in the LAC, all believed to be native to Latin America, the Caribbean region, or eastern USA.

Early recognition of symptoms of infection is essential to prevent diseases from spreading through nurseries and into planted systems. In plantations, symptoms of Ceratocystis manginecans infection are typically wilt and canker but symptoms may differ at the nursery stage and as yet have not been described for Eucalyptus pellita. In this study, disease symptoms were recorded over a three-month period in an E. pellita nursery and isolates trapped. Then, to clearly identify those relating to ceratocystis disease and any related mortality, two C. manginecans isolates were re-inoculated by either spraying or dipping of healthy plantlets raised by tissue culture and mini-cuttings. Many disease symptoms were observed in the nursery but only perithecia on the leaves and stems was a clear indication of the presence of a Ceratocystis pathogen. Nine pathogenic fungal species, including C. manginecans, were isolated and identified by DNA analysis. Koch’s postulates indicated that leaf blight associated with C. manginecans was black-brown in colour, irregular, with uneven edges and surrounded by a yellow margin or halo on the upper leaf surface. Mortality following inoculation of plantlets by spraying immediately after planting was > 70%; by contrast, mortality of mini-cuttings was < 50% if spraying was done just before transplanting but < 20% if done 7 or 14 days after transplanting. Isolate EP313C caused significantly greater mortality than isolate EP106C. The research showed that C. manginecans and other pathogens were present in the nursery environment and must be identified and managed as part of good nursery practice.

The deployment of Eucalyptus pellita trees that are resistant to Ceratocystis manginecans is essential for the commercial plantations and therefore the sustainability of forest industries in Southeast Asia that utilize this resource. Current screening procedures are time-consuming and expensive but could be expedited with the aid of marker-assisted selection and breeding. The identification of genotypes with resistance to the disease may be facilitated if microsatellite markers developed in other Eucalyptus species are transferable and can be linked to quantitative trait loci (QTL) for disease resistance. This possibility was tested in 111 full-sib progenies and their parents by genotyping with 49 microsatellite markers developed in other Eucalyptus species. Disease development was assessed after stem inoculation with C. manginecans isolate Am60C. The disease index (DI) varied from 0 to 20% of stem length. There was a continuous distribution of resistant and susceptible seedlings with 60% in the resistant category. Of the 30 acceptable markers, 17 (56%) defined two linkage groups (LG). In each LG, one QTL with a significant logarithm of odds (LODs &gt; 13) was identified. The transferability of microsatellite markers developed in other Eucalyptus species facilitated the rapid identification of LGs and QTLs in E. pellita. To further refine the linkage map, the testing of more microsatellite markers and a larger population of progenies are required.

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.

Phytochemical Identification and Comparative Antifungal Efficacy of Dodonaea viscosa L. Fractions against Ceratocystis manginecans

First report of canker and wilt disease in eucalypt caused by Ceratocystis manginecans in Vietnam

The planted forest area in Vietnam increased from 3.0 to 4.4 million hectares in the period 2010–2020, but the loss of productivity from pests and diseases continues to be a problem. During this period, frequent and systematic plantation forest health surveys were conducted on 12 native and 4 exotic genera of trees as well as bamboo across eight forest geographic regions of Vietnam. Damage caused by insects and pathogens was quantified in the field and laboratory in Hanoi. The threats of greatest concern were from folivores (Antheraea frithi, Arthroschista hilaralis, Atteva fabriciella, Hieroglyphus tonkinensis, Lycaria westermanni,Krananda semihyalina, and Moduza procris), wood borers (Batocera lineolata, Euwallacea fornicatus, Tapinolachnus lacordairei, Xyleborus perforans, and Xystrocera festiva), sap-sucking insects (Aulacaspis tubercularis and Helopeltis theivora) and pathogens (Ceratocystis manginecans, Fusarium solani, and Phytophthora acaciivora). The number of new and emerging pests and pathogens increased over time from 2 in 2011 to 17 in 2020, as the damage became more widespread. To manage these pests and diseases, it is necessary to further invest in the selection and breeding of resistant genotypes, improve nursery hygiene and silvicultural operations, and adopt integrated pest management schemes. Consideration should be given to developing forest health monitoring protocols for forest reserves and other special-purpose forests.

Species in the Ceratocystis manginecans complex are important fungal pathogens of plantation trees globally. The most important hosts include species of Eucalyptus, Acacia, Mangifera, and Punica. Despite their relevance and widespread occurrence, little is known regarding their population genetics and how this might relate to their host associations or geographic regions in which they occur. A global collection of 491 isolates representing the C. manginecans complex, from four different plant hosts and nine countries, were genotyped using microsatellite markers. Population genetic analyses using numerous tools were conducted to interrogate how their genetic diversity and structure might be affected by host or areas of occurrence. Results of genetic diversity studies showed that when grouping isolates into populations based on their host associations, the population on Eucalyptus was most diverse, and it also has a broad global distribution. When considering countries of origin as a basis for defining populations, the gene and genotypic diversity were highest in populations from China, Indonesia, and Brazil. In contrast, populations from Oman and Pakistan collected from Mangifera had the lowest genetic diversity and were clonal. Molecular variance, population differentiation, and network and structure analyses showed that the genetic structure of isolates in the C. manginecans complex is influenced by both host association as well as geographical isolation. Furthermore, the results reflected the movement of genotypes between plant hosts and geographic regions that have implications regarding the broad global distribution of this pathogen.

Pathogenicity of Ceratocystis manginecans in inoculated Acacia roots

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.

Ceratocystis manginecans causes wilt and death of plants in several important crops and native vegetation in Indonesia. Ceratocystis wilt was recently found to be causing substantial mortality in bullet wood (Mimusops elengi) in South Sumatra. The aim of this study was to describe the symptomatology of the new disease and characterise isolates of C. manginecans obtained from bullet wood plants. Affected plants showed substantial discoloration of the woody xylem and wilt-type symptoms of the foliage, with the eventual death of the whole plant. Isolations from infected trees yielded a fungus that was similar morphologically to C. manginecans, with typical hat-shaped ascospores and light-coloured perithecial bases. Sequencing of the internal transcribed spacer (ITS) and ?-tubulin of isolates confirmed their identification, grouping them with C. manginecans and separating them from all other Ceratocystis species. This is the first report of C. manginecans in Indonesia causing wilt and death on bullet wood. C. manginecans is an important pathogen, and strategies to reduce losses need to be established in Indonesia because the aggressiveness of C. manginecans to bullet wood has been shown in inoculation experiments

Eucalyptus pellita F. Muell. has become an important tree species in the forest plantations of SE Asia, and in Malaysian Borneo in particular, to replace thousands of hectares of Acacia mangium Willd. which has suffered significant loss caused by Ceratocystis manginecans infection in Sabah, Malaysia. Since its first introduction at a commercial scale in 2012, E. pellita has been planted in many areas in the region. The species replacement requires new silvicultural practices to induce the adaptability of E. pellita to grow in the region and this includes relevant research to optimise such regimes as planting distance, pruning, weeding practices and nutrition regimes. In this present study, the nutritional status of the foliage was investigated with the aim to develop near infrared spectroscopic calibrations that can be used to monitor and quantify nutrient status, particularly total foliar nitrogen (N) and phosphorus (P) in the field. Spectra acquired on fresh foliage in situ on the tree could be used to predict N and P with accuracy suitable for operational decision-making regards fertiliser application. If greater accuracy is required, spectra acquired on dry, milled foliage could be used to predict N and P within a relative error of 10% (R2c, r2CV, RMSEP, RPD = 0.77, 0.71, 0.02 g 100 g-1, 1.9 for foliar P and = 0.90, 0.88, 0.21 g 100 g-1, 3.0 for foliar N on dry, milled foliage). The ultimate application of this is in situ nutrient monitoring, particularly to aid longitudinal studies in fertiliser trial plots and forest operations, as the non-destructive nature of NIR spectroscopy would enable regular monitoring of individual leaves over time without the need to destructively sample them. This would aid the temporal and spatial analysis of field data.

Terpenes represent the biggest group of natural compounds on earth. This large class of organic hydrocarbons is distributed among all cellular organisms, including fungi. The different classes of terpenes produced by fungi are mono, sesqui, di- and triterpenes, although triterpene ergosterol is the main sterol identified in cell membranes of these organisms. The availability of genomic data from members in the Ceratocystidaceae enabled the detection and characterization of the genes encoding the enzymes in the mevalonate and ergosterol biosynthetic pathways. Using a bioinformatics approach, fungal orthologs of sterol biosynthesis genes in nine different species of the Ceratocystidaceae were identified. Ergosterol and some of the intermediates in the pathway were also detected in seven species (Ceratocystis manginecans, C. adiposa, Huntiella moniliformis, Thielaviopsis punctulata, Bretziella fagacearum, Endoconidiophora polonica and Davidsoniella virescens), using gas chromatography-mass spectrometry analysis. The average ergosterol content differed among different genera of Ceratocystidaceae. We also identified all possible terpene related genes and possible biosynthetic clusters in the genomes used in this study. We found a highly conserved terpene biosynthesis gene cluster containing some genes encoding ergosterol biosynthesis enzymes in the analysed genomes. An additional possible terpene gene cluster was also identified in all of the Ceratocystidaceae. We also evaluated the sensitivity of the Ceratocystidaceae to a triazole fungicide that inhibits ergosterol synthesis. The results showed that different members of this family behave differently when exposed to different concentrations of triazole tebuconazole.

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.

Ceratocystis manginecans causes mango wilt with significant economic losses. In the infection court, cerato-platanin (CP) family proteins (CPPs) are believed to involve in pathogenesis but has not been determined in C. manginecans. To confirm this function, a CP protein (CmCP) of C. manginecans was characterized in this study. A protoplast of C. manginecans was prepared by treating its mycelia with driselase and lysing enzymes. The cmcp gene was edited using CRISPR/Cas-U6-1 expression vectors in 60% PEG and 50 μg/mL hygromycin B in the medium, resulting in mutants with cmcp deletion (Δcmcp). A complemented mutant (Δcmcp-C) was obtained by transforming cmcp to Δcmcp. Both Δcmcp and Δcmcp-C were characterized by comparing them with a wild-type strain on morphology, mycelial growth, conidial production and pathogenicity. Additionally, cmcp was transformed and expressed in Pichia pastoris, and the derived recombinant protein CmCP caused a severe necrosis on Nicotiana tabacum leaves. CmCP-treated plant leaves showed symptoms of hypersensitive response including electrolyte leakage, reactive oxygen species generation and overexpression of defense-related genes PR-1, PAD3, ERF1, HSR203J, and HIN1. All those results suggested that cmcp gene was required for the growth development of C. manginecans and functioned as a major pathogenicity factor in mango infection.