First report of Eucalyptus wilt caused by Chalaropsis thielavioides in China

Eucalyptus species are widely planted in the tropics and subtropics, and eucalyptus is among the most important cash crop in Southern China. One of the most important diseases on eucalyptus is Ceratocystis wilt, caused by the fungus Ceratocystis fimbriata Ellis & Halst., and the genus name Chalaropsis has been proposed for anamorphs of Ceratocystis species (de Beer et al. 2014). During April 2018, severely infected Eucalyptus robusta trees were observed in Kunming, Yunnan Province, China. Symptomatic trees initially exhibited yellowing and wilting of foliage on individual branches, then spread to the whole canopy, sometimes followed by death of the whole tree. Reddish-brown to dark-brown discoloration in the woody xylem of affected trees, sometimes a grayish white layer of fungal growth may be seen. The disease was observed on 16% of trees surveyed. The base of trunks with typical symptoms were collected, then the discolored xylem tissues were surface disinfected with 75% ethanol for 30 s and 0.1% mercuric chloride (HgCl2) solution for 2 min, rinsed three times with sterile distilled water, plated onto potato dextrose agar (PDA) medium, and incubated at 25°C. After 6 days, a fungus was consistently observed growing from the tissue. Three isolates were obtained. In culture, colonies reaching 54mm diam within 15 days, mycelium initially white, then becoming celadon. Endoconidia unicellular, smooth, cylindrical, straight, biguttulate, 11.21 - 32.26 × 4.12 - 5.25 μm. Phialides produced on short, septate, aerial hyphae, lageniform and chain of phialoconidia (3.62 - 5.89 × 31.39 - 65.76 μm) were also observed. Chlamydospores (11.45 - 14.26 × 10.06 - 12.22 μm) were single, dark, thick-walled. Morphological characteristics of the fungus were consistent with the description of Chalaropsis thielavioides (Paulin-Mahady et al. 2002). The two of three isolates were used for molecular identification and genomic DNA was extracted from isolates (EKY2-2-1, EKY2-2-2) using the chelex-100 method (Xu et al. 2020). The ITS region of rDNA was sequenced using the procedures of Thorpe et al. (2005). Analysis of ITS sequence data (GenBank accessions MW242701, MW242702) showed that the isolates were 99% - 100% homologous to isolates of C. thielavioides from Hevea rubber, Monstera deliciosa L. and ants in China and Rosa sp. in Australia (GenBank accessions KT963172, KJ511482, KT963173 and KX954598) by BLAST analysis. Neighbor-joining (NJ) phylogenetic analysis were performed using MEGA 6.06 based on ITS sequences (Fig 1), the evolutionary distances were computed using the Maximum Composite Likelihood method. Analyses showed that both isolates (EKY2-2-1, EKY2-2-2) located on the same clade with all C. thielavioides, and clustered with the C. thielavioides strains with high bootstrap support (97% - 100%). Therefore, the fungus was identified as C. thielavioides based on morphology and molecular evidences. Pathogenicity of C. thielavioides was tested by inoculation of six one-year-old pot grown Eucalyptus citriodora seedlings. The sterilized soil of six seedlings was inoculated by drenching with 20 ml spore suspension (2.0 × 106 spores / ml). Control plants were inoculated with 20 ml of sterile distilled water. The seedlings were kept in a controlled greenhouse at 25°C and watered weekly. After one month incubation, all the isolates produced wilt symptoms, whereas control trees showed no symptoms. The original fungus was successfully re-isolated from inoculated trees and identified as C. thielavioides according to the methods described above, and no fungal growth was observed in the controls, thus satisfying Koch's postulates. Although wilt and canker caused by Ceratocystis fimbriata on eucalyptus have been previously reported in Brazil, Uruguay, Uganda, China and Pakistan (Ferreira et al. 1999; Li et al. 2014; Alam et al. 2017), eucalyptus wilt caused by C. thielavioides has not been reported anywhere. Also, wilt of rubber tree and postharvest rot on carrot caused by C. thielavioides have been reported (Li et al. 2021; Xu et al. 2020). To our knowledge, this is the first report of eucalyptus wilt caused by C. thielavioides in China.

First Report of Coffee Canker Disease Caused by Ceratocystis fimbriata in China

Coffee (Coffea arabica L.) is one of the most important agricultural commodities in the world market. As an important cash crop in China, coffee is cultivated mainly in Yunnan and Hainan provinces. During October 2013 and September 2020, coffee trees showing typical dieback and wilt symptoms were found in Nanping town (N 22° 38', E 101° 0'), Pu’er, and Puwen town (N 22° 32', E 101° 4'), Xishuangbanna in Yunnan province, China. Symptomatic trees initially exhibited yellowing of foliage, expanding in size along the leaf margin, then became blighted and dry, and the internal xylem in main stem discolored brown to black. Infected trees eventually developed dieback and wilt. Disease incidence ranged from 10% to 22% and 25% to 40% of crown symptoms in the affected coffee trees. In extreme cases, 50% out of 380 trees were affected. The stems of coffee trees with typical symptoms were collected, and then the diseased tissues were surface disinfected with 75% ethanol for 30 s and 0.1% mercuric chloride (HgCl2) solution for 2 min, rinsed three times with sterile distilled water, plated onto potato dextrose agar (PDA) medium, and incubated at 25°C. After 6 days, fungal mycelium was observed growing from the tissue. Three isolates (C3-1, C3-2, and C3-2-1) were obtained by picking spore masses from the apices of perithecia and transferring them to malt extract agar (MEA) medium and incubated at 25°C for 10 days to observe the cultural features. In culture, colonies reaching 65 mm within 10 days, mycelium initially white, then becoming light blue-green. After 6 days of formation, perithecia were black, globose (123.8 - 173.4 μm × 138.2 - 180.6 μm), and showed a long black neck (414.2 - 650.0 μm). Ascospores with outer cell wall forming a brim, hat-shaped, accumulating in a mucilaginous mass at the tips of ostiolar hyphae (4.3 μm × 6.0 μm). Cylindrical endoconidia (14.1 - 45.2 μm × 3.5 - 5.7 μm) were hyaline. Chain of barrel-shaped conidia (6.6 - 10.2 μm × 6.8 - 8.8 μm) were found. Aleuroconidia (10.8 - 16.9 μm × 9.1 - 13.0 μm) were olive-brown, ovoid or obpyriform, and smooth. Morphological characteristics of the fungus were consistent with the description of Ceratocystis fimbriata Ellis & Halst. (Engelbrecht and Harrington 2005). The three isolates were used for molecular identification, and their genomic DNA was extracted using the chelex-100 method (Xu et al. 2020). The internal transcribed spacer (ITS) region of rDNA was sequenced using the procedures of Thorpe et al. (2005). Analysis of the ITS sequence data (GenBank accessions KY580836, KJ511480, and KJ511479) showed that the isolates were 100% homologous to isolates of C. fimbriata from Punica granatum, Camellia sinensis, and Cucumis sativus in China (GenBank accessions KY580891, KY580870, and MH535909, respectively) by BLAST analysis. Neighbor-joining (NJ) phylogenetic analysis was performed using MEGA 6.06 based on the ITS sequences. The three isolates were clustered on the same clade with other C. fimbriata isolates with a high bootstrap value (90%). Therefore, the fungus was identified as C. fimbriata based on both morphological and molecular characteristics. Pathogenicity of the three isolates was tested by inoculating one-year-old pot grown coffee seedlings (C. arabica) through drenching the loams with 30 ml spore suspension (1 × 106 spores/ml). Control plants were inoculated with 30 ml of sterile distilled water. The trees were kept in a controlled greenhouse at 25°C and watered weekly. One month after inoculation, all inoculated plants produced typical dieback and wilt symptoms, whereas the control trees showed no symptoms. The same fungus was isolated from the inoculated trees on PDA and identified as C. fimbriata according to the methods described above, and no fungal growth was observed in the controls, thus fulfilling the Koch's postulates. Coffee canker disease caused by C. fimbriata has been reported in Indonesia and Colombia (Marin et al. 2003). To our knowledge, this is the first report of C. fimbriata causing canker disease of coffee trees in China.

First report of rubber tree wilt caused by Ceratocystis fimbriata in China

The rubber tree (Hevea brasiliensis) is an important economic resource for the rubber and latex industry. During November 2013 and June 2016, rubber trees showing typical wilt symptoms were found in Mengla, Xishuangbannan, Yunnan, China (N 21° 28', E 101° 33'). Symptomatic trees initially exhibited wilting of foliage on individual branches, then spread to the whole canopy, finally followed by death of the whole tree. Dark-blue to black discoloration was observed in the inner bark and affected xylem, a grayish layer of fungal growth and sporulation occasionally. The disease was detected on 20% of trees surveyed. The diseased tissues of three rubber trees were surface disinfected with 75% ethanol for 30 s and 0.1% mercuric chloride (HgCl2) for 2 min, rinsed three times with sterile distilled water, plated onto potato dextrose agar (PDA), and incubated at 25°C. After 7 days, a fungus was consistently observed growing from the tissue. Three single-spore isolates were obtained. In culture, colonies reaching 69 mm diam within 10 days, mycelium was initially white, then becoming celadon. After 5 days of perithecium formation, observed perithecia were black, globose (173.1 - 237.9 × 175.6 - 217.2 μm) and showed a long black neck (507.3 - 794.1 μm). Ascospore with outer cell wall forming a brim, hat-shaped at the tips of ostiolar hyphae (3.43 × 5.63 μm). Cylindrical endoconidia (10.5 - 39.7 × 3.5 - 6.6 μm) were hyaline. Chain of barrel-shaped conidia (7.2 - 9.5 × 4.1 - 6.2 μm) was found. Aleuroconidia were ovoid or obpyriform, and smooth (10.2 - 14.1 × 8.4 - 10.6 μm). Morphological characteristics of the fungus were consistent with the description of Ceratocystis fimbriata (Engelbrecht and Harrington 2005). The genomic DNA was extracted from isolates (XJm10-2-5, XJm8-2-5, XJm4) using the Chelex-100 method (Xu et al. 2020). The ITS region of rDNA was sequenced using the procedures of Thorpe et al. (2005). Analysis of ITS sequence data (GenBank accessions KJ511488, KJ511485, KT963149) showed that the isolates were 100% homologous to those of the isolates on Punica granatum and Colocasia esculenta from China (GenBank accessions KT963152, MH793673) by BLAST analysis. Neighbor-joining phylogenetic analyse were performed using MEGA 6.06 based on ITS sequences (Fig. 1). Analyses showed that all isolates located on the same clade with all C. fimbriata with a high bootstrap support. Therefore, the fungus was identified as C. fimbriata based on morphology and molecular evidences. Pathogenicity of C. fimbriata isolated from this study was tested by inoculation of three one-year-old pot-grown (3L) seedlings of rubber tree. The soil of three seedlings was inoculated by drenching with 30 ml spore suspension (2.0 × 106 spores / ml). Three control plants were inoculated with 30 ml of sterile distilled water. The experiment was repeated three times. The plants were kept in a controlled greenhouse at 25°C and watered weekly. After the inoculation for one month, all the isolates produced typical wilt symptoms, while control plants showed no symptoms. The original fungus was successfully re-isolated from inoculated trees and identified as C. fimbriata according to the methods described above. The pathogenicity assay showed that C. fimbriata was pathogenic to rubber trees. C. fimbriata was first reported on rubber tree in Brazil (Albuquerque et al. 1972; Silveira et al. 1985). To the best of our knowledge, this is the first report of C. fimbriata causing wilt of rubber tree in China. This finding contributes to understanding the diversity of this pathogen, and it appears to be a significant threat to rubber trees in its ecosystem.

Survey, Identification, and Pathogenicity of Ceratocystis fimbriata Complex Associated with Wilt Disease on Acacia mangium in Malaysia

Ceratocystis wilt disease surveys were conducted in three selected Malaysian Acacia mangium plantations. These completed surveys revealed the occurrence of the wilt disease, with the incidence of infection ranging from 7.5% to 13.6%. Signs of wood-boring insects, bark peeling due to squirrel activity, and pruning wounds were often associated with this disease. The fungus most frequently isolated from the diseased trees was the Ceratocystis fungus. The analysis on the morphological characteristics has identified the fungus as Ceratocystis fimbriata complex. Phylogenetic analysis based on the sequences of the ITS, and concatenated sequences of EF1α-βT regions grouped the isolates within the C. fimbriata sensu stricto, in comparison to other C. fimbriata isolates. Pathogenicity tests were conducted on six to nine-month-old healthy A. mangium seedlings by inoculating these seedlings with eight out of the 16 isolates. The results demonstrated that all the isolates were pathogenic, with mortality beginning as early as two weeks after inoculation. However, an ANOVA test indicated a significant difference between the pathogenicity levels among the fungal isolates. The results also showed that pathogen aggressiveness was not correlated with geographical origin. A host range test was also conducted by using C. fimbriata SSB3 and FRIM1162 isolates against several forest plantation species. The findings suggested that only A. mangium was susceptible to C. fimbriata. The other species remained healthy with no symptoms of infection even after seven weeks of treatment, as compared to the A. mangium species, where between 38 to 60% of the inoculated plants had died. This study provides new information on the status of Ceratocystis wilt disease, especially on the occurrence and effects on A. mangium plantation, by giving insights on how to control and manage this ferocious plant pathogen in the future.

First record of Ceratocystis fimbriata causing quick wilt of Calotropis gigantea in Pakistan

Calotropis gigantea belongs to the family Asclepiadaceae, native to Asia and commonly known as crown flower. C. gigantea was identified as an important medicinal plant in Asia and also harvested to obtain the fibres from the stem (Ganeshan et al. 2018; Narayanasamy et al. 2020). In April 2021, a quick wilt epidemic of C. gigantea was observed in District Gujranwala (32°05'58.0"N 74°02'38.0"E) Punjab, Pakistan. The wilt symptoms were very severe on mature plants with 60% disease incidence. Affected plants exhibited yellowing and quick wilting of leaves on each branch that eventually spread to the whole plant (Figure 1). Black patches were observed on the stem bark and cross section of infected stem showed black streaks in the xylem. The samples (n=33) were collected from symptomatic plants. Infected tissues were excised into 4-5mm segments, surface disinfested with 1% NaClO and rinsed 3 times with sterilized distilled water. To induce the sporulation of the fungus, the described carrot baiting method of Moller et al., (1968) was used. Single ascoma from carrot baits were picked under a microscope and transferred to malt extract agar (MEA) medium and incubated at 25°C. Purified cultures were obtained by single spore culture method and all the isolates were preserved with 35% glycerol at -80°C. The fungal colony was greyish olive with overripe banana odour. Hyphae were smooth and segmented. The perithecia were globose, black, measuring 190.27 to 257.34 × 186.11 to 243.24 µm, and showed a long black neck (732 to 977.34 µm). Cylindrical conidia (9.46 to 22.97 µm × 2.7 to 5.41 µm), barrel-shaped conidia (6.34 to 7.95× 8.15-10.41 µm) and chlamydospores (7.65-11.38µm×10.17-16.81µm) were observed (Figure 2). The isolates were identified as Ceratocystis fimbriata based on morphology and similar results were reported by Engelbrecht et al. (2005) and Suwandi et al. (2021). Genomic DNA was extracted from all isolates and multi-locus sequence analysis approach was used for molecular identification. The Internal Transcribed Spacers (ITS) region, the Translation Elongation Factor 1-α gene (TEF) and β-tubulin gene (TUB) were amplified using ITS1/4 (White et al. 1990), EF1-728F/ EF1-986R (Carbone et at. 1999) and βt1a/βt1b (Glass and Donaldson, 1995). Based on the BLAST analysis, all isolates were identified as C. fimbriata. The sequences of the representative isolate AK-W17 were submitted to the GenBank, NCBI database with the accession numbers (ITS:MZ711226), (TEF: MZ714595) and (TUB:MZ714596) showing 100% similarity with AF395687(ITS), MG980731(TEF) and AY177227(TUB) accessions of C. fimbriata. Based on Sequences similarity, representative isolate AK-W17 grouped with the isolates which representing C. fimbriata. Pathogenicity test was conducted on healthy C. gigantea seedlings grown in pots. A fungal mycelium plug (4×4mm) from 15 days old culture of AK-W17 was inserted into a slit (5 × 8 mm and made by puncturing the bark surface) in the stem bark, and the artificial wound was covered with wetted tissue paper and secured with Parafilm to maintain humidity, while control treatment was inoculated with only MEA medium plug. The seedlings were incubated under 70% relative humidity (RH) and 28°C in greenhouse. After 24 days, the inoculated seedlings showed 100% wilting identical to the original plant from which they were isolated (Figure 3). The control treatment had no symptoms. The pathogenic fungus was reisolated and identifies as C. fimbriata based on morphological and molecular characterization. C. fimbriata has been also reported to cause wilt disease in Dalbergia sissoo (Harrington et al. 2015) and pomegranate (Alam et al. 2017) in Pakistan. To our knowledge, this is the first report of C. fimbriata to cause C. gigantea quick wilt in Pakistan. C. fimbriata is one of the most aggressive plant pathogens and rapidly spread worldwide, so it is critical to implement appropriate management practices to reduce the fungus attack on plants.

PROPERTIES OF TEAK WOOD INFECTED BY Ceratocystis fimbriata

In Brazil, the growth of Tectona grandis (teak) plantations is accompanied by an increased incidence of Ceratocystis wilt, a fungal disease that colonizes the vascular system of such tree. The objective of this work was to evaluate the properties of teak wood infected with Ceratocystis fimbriata at different radial positions. Ten 17-year-old trees (five infected and five healthy) were collected. A disk was removed from the base of each tree to determine Janka hardness, basic density, anatomical analysis, colorimetric parameters in the CIEL*a*b* system and sodium hydroxide solubility (NaOH). There were no significant differences for radial position and health for basic density and Janka hardness of the wood, with mean values of 0.488 g.cm3 and 58.66 MPa, respectively. The deposition of dark compounds was observed inside the parenchyma cells and fibers, as well as the formation of tyloses in the sapwood. Yellow pigment was predominant in the color formation of teak wood, which was influenced by radial position, with the sapwood being lighter. Regarding C. fimbriata infection, the color change was more evident in sapwood. Conversely, the NaOH solubility was lower for the sapwood attacked by the fungus. In conclusion, the changes in teak wood infected by C. fimbriata are visual and the anatomical structures do not deteriorate.

Predicting Mango Sudden Decline Due to Ceratocystis fimbriata Under a Changing Climate

Mango fruit trees are an important fruit crop due to their high value. Mango sudden decline (MSD) is a major disease that threatens mango trees in Oman and worldwide. The objective of this study was to identify those areas in northern Oman in which Ceratocystis fimbriata (a plant fungal pathogen causing MSD) may establish itself under various climate change scenarios. The MaxEnt model used in this study was based on data for the period 1970-2000 and then projected to future climate periods. This study modeled the future distribution of C. fimbriata for 2021–2040, 2041–2060, 2061–2080, and 2081–2100 climatic scenarios. Fifteen affected locations and seven bioclimatic variables were investigated in this study. The model showed values between 0.896 and 0.913 (habitat suitability) which represented a good model outcome. The jackknife test showed that the mean diurnal range in temperature, precipitation of the driest month, and elevation contributed to C. fimbriata distribution. From 2021 through 2040, a total area of 1,889 km2 was found to be highly suitable for C. fimbriata in Northern Oman. Compared with the 2021–2040 period, the poorly suitable area would increase in both 2041–2060 and 2081–2100 periods. The moderately suitable regions for C. fimbriata would decrease under all scenarios investigated. However, the total area of the suitable areas, with all scenarios, would increase, except during the 2041-2060 period. This research offers a tool to better manage and prevent the possible Ceratocystis blight (C. fimbriata) and bark beetle (Hypocryphalus mangiferae) invasions under future projected climatic scenarios. Keywords: Mango sudden decline (MSD), “Ceratocystis fimbriata”, bioclimatic variables, climate change, Sultanate of Oman, Maxent.