Eucalyptus spp., most of which are native to Australia, are one of the world's most commonly planted forestry crops, and have been widely planted in the tropics and subtropics. Eucalyptus trees are distributed in more than 600 counties in 19 provinces/regions throughout China, especially in the southern regions of the country (1). From April of 2013 to May of 2014, eucalyptus trees were noted to be wilting and dying rapidly in Kunming, Yunnan Province (25°02′ N, 102°42′ E), southwest China. Symptomatic trees typically have many wilted or dead leaves hanging throughout the tree or on some major branches, sometimes followed by death of the whole tree. Dark brown to black discoloration can be seen in the woody xylem, and elongated cankers were also present on some affected trees. A fungus was consistently isolated from the leaves and cankers when symptomatic tissue was incubated between two slices of fresh carrot root. Spore masses were removed from the apices of perithecia, transferred to malt extract agar medium (MEA), and incubated at 25°C. Perithecia developed on the medium, and were black, globose, 212.5 to 242.4 × 207.5 to 254.2 μm, and possessed a long black neck (952.3 to 1,303.3 μm). Ascospores had the typical “hat” morphology and were 4.3 to 5.2 × 3.0 to 3.8 μm. Cylindrical endoconidia (11.2 to 22.2 × 3.9 to 6.1 μm) were found. Chlamydospores produced on media were ovoid and smooth, and were 7.8 to 9.7 × 9.9 to 12.8 μm. Chains of barrel-shaped conidia were not found. PCR amplification and sequencing of the ITS region of rDNA were carried out for one isolate, E2-2, using the procedures of Thorpe et al. (3). Analysis of ITS sequence data (GenBank Accession No. KJ511481) showed that the isolates were 99% homologous to the isolate of C. fimbriata from diseased Colocasia esculenta in Cuba, China, and Hawaii (AY526304 to 06) by BLAST analysis. Thus, the fungus was identified as C. fimbriata based on morphological and molecular characteristics. Pathogenicity tests were made on 1-year-old E. grandis seedlings as follows. A conidial suspension of each isolate was diluted to 106 conidia/ml, and 0.2 ml was injected into wounds on three petioles on each of five healthy plants of E. grandis, and control seedlings were injected with sterile water that had been placed on MEA plates. The seedlings were incubated at 25°C in moist chambers. After 3 days, all inoculated E. grandis plants showed dark brown to black discoloration in the leaf axils. After 5 days, leaf wilting was present. C. fimbriata was re-isolated from all inoculated symptomatic tissue. No symptoms were visible in the control plants and no fungus was isolated from them. Ceratocystis wilt was first observed in eucalyptus in 1997 in the state of Bahia. This was followed by a report of C. fimbriata causing wilt of E. grandis in the Republic of Congo, Uganda, and Uruguay (2). Chen et al. reported two species of Ceratocystis, C. acaciivora and a previously undescribed species C. chinaeucensis, from eucalyptus plantations in Guangdong Province in China (1). To our knowledge, this is the first report of C. fimbriata causing wilt of eucalyptus in China. References: (1) S. F. Chen et al. Fungal Diversity 58:267, 2011. (2) F. A. Ferreira et al. Fitopatol. Bras. 24:284, 1999. (3) D. J. Thorpe et al. Phytopathology 95:316, 2005.

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Rubber tree (Hevea brasiliensis (Willd. ex Adr. Juss) Müll. Arg.) is used for the extraction of natural rubber and is an economically and socially important estate crop commodity in many Asian countries such as Indonesia, Malaysia, Thailand, India, Sri Lanka, China and several countries in Africa (Pu et al, 2007). Xishuangbanna City and Wenshan City are the main rubber cultivation areas in Yunnan Province, China. In November 2012, rubber tree showing typical wilt symptoms (Fig. 1 A) and vascular stains (Fig. 1 B) were found in Mengla County, Xishuangbanna City. This disease was destructive in these trees and plant wilt death rate reached 5%. The diseased wood pieces (0.5cm long) from trunk of rubber was surface disinfected with 75% ethanol for 30s and 0.1% mercuric chloride (HgCl2) for 2min, rinsed three times with sterile distilled water, plated onto malt extract agar medium (MEA), and incubated at 28℃. After 7 days, fungal-like filaments were growing from the diseased trunk. Six cultures from 6 rubber trunk were obtained and incubated on MEA at 28℃, after 7 days to observe the cultural features. The mycelium of each culture was white initially on MEA, and then became dark green. Cylindrical endoconidia apices rounded, non-septate, smooth, single or borne in chains (8.9 to 23.6 × 3.81 to 6.3μm) (Fig. 1 C). Chlamydospores (Fig. 1 D) were abundant, thick walled, smooth, forming singly or in chains (11.1 to 19.2 × 9.4 to 12.0μm). The mould fungus was identifed as Chalaropsis based on morphology (Paulin-Mahady et al. 2002). PCR amplification was carried out for 3 isolates, using rDNA internal transcribed spacer (ITS) primer pairs ITS1F and ITS4 (Thorpe et al. 2005). The nucleotide sequences were deposited in the GenBank data base and used in a Blast search of GenBank. Blast analysis of sequenced isolates XJm8-2-6, XJm8-2 and XJm10-2-6 (accessions KJ511486, KJ511487, KJ511489 respectively) had 99% identity to Ch. thielavioides strains hy (KF356186) and C1630 (AF275491). Thus the pathogen was identified as Ch. thielavioides based on morphological characteristics and rDNA-ITS sequence analysis. Pathogenicity test of the isolate (XJm8-2) was conducted on five 1-year-old rubber seedlings. The soil of 5 rubber seedlings was inoculated by drenching with 40 ml spore suspension (106 spores / ml). Five control seedlings were inoculated with 40 ml of sterile distilled water. All the seedlings were maintained in a controlled greenhouse at 25°C and watered weekly. After inoculated 6 weeks, all the seedlings with spore suspension produced wilt symptoms, as disease progressed, inoculated leaves withered (Fig. 1 E) and vascular stains (Fig. 1 F) by 4 months. While control seedlings inoculated with sterile distilled water remained healthy. The pathogen re-isolated from all inoculated symptomatic trunk was identical to the isolates by morphology and ITS analysis. But no pathogen was isolated from the control seedlings. The pathogenicity assay showed that Ch. thielavioides was pathogenic to rubber trees. Blight caused on rubber tree by Ceratocystis fimbriata previously in Brazil (Valdetaro et al. 2015), and wilt by Ch. thielavioides was not reported. The asexual states of most species in Ceratocystis are “chalara” or “thielaviopsis” (de Beer et al. 2014). To our knowledge, this is the first report of this fungus causing wilt of rubber in China. The spread of this disease may pose a threat to rubber production in China.

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