First Report of Black Root Disease Caused by Chaetomium globosum on Rubber Tree Seedlings in Yunnan, China

Abstract

In September 2016, a new disease of the root tips of rubber tree seedlings (Hevea brasiliensis Mull Arg.) was found in Jinghong City, Yunnan, China. Diseased seedlings had stunted, blackened, but not rotten root tips. The disease incidence at the seedbeds could reach as high as 98%. Some stems of the seedlings developed lesions or cankers, but their leaves were free of any disease symptoms. Samples of infected root tips were washed with tap water, dried, and cut into 0.2- to 0.3-cm pieces and then surface sterilized with 0.1% mercuric chloride solution for 30 s followed by three 1-min rinses with sterilized water. The pieces near the edge of the symptomatic tissues were placed on potato dextrose agar (PDA) medium and incubated at 25°C for 5 days. Such infected tissues consistently yielded a filamentous fungus with similar cultural and morphological characteristics on PDA. A representative pure culture designated as strain XJ160930 was finally obtained after several isolation transfers to PDA using a single ascospore. Strain XJ160930 had the following culture characteristics: A colony plug on 7-day PDA culture at 25°C exhibited an entire edge, with sparse white to olivaceous buff aerial hyphae when young, then without aerial hyphae and becoming citrine greenish olivaceous to gray olivaceous with fulvous, apricot to sienna exudates, or olivaceous to olivaceous gray exudates diffusing into the medium. Ascomata superficial, ostiolate, greenish olivaceous or slightly dark olivaceous buff to gray or dull green in reflected light owing to ascomatal hairs, subglobose, ovate or obovate, 110 to 240 μm diameter, and 160 to 410 µm high. Ascomatal wall brown, textura intricata in surface view. Terminal hairs finely warty, brown, erect or undulate to loosely coiled with erect or flexuous lower part, tapering and fading toward the tips, 2 to 5 μm diameter near the base. Lateral hairs brown, flexuous, fading and tapering toward the tips. Ascospores brown when mature, limoniform, usually biapiculate, bilaterally flattened, 7 to 8.5 × 5.5 to 7 μm, with an apical germ pore. Based on these morphological characteristics, the fungus was identified as Chaetomium globosum (Wang et al. 2016). To determine the pathogenicity, isolate XJ160930 was inoculated onto GT1 rubber tree seeds. Thirty seeds each were sowed in three 30-cm pots of clean sand, and 500 ml of spore suspension (1.0 × 10⁶ conidia/ml) prepared from 10-day-old culture on PDA was sprayed onto these pots. Water was used as a control treatment. All the pots were placed in a greenhouse at approximately 25 to 28°C and sprayed with sterilized water once every 3 days. After 20 days of incubation, the seeds began to germinate and grow. When these seedlings were pulled out then, all those from the fungus-inoculated pots showed root-tip stunts and darkening, like the symptoms observed in the field-infected seedlings. The pathogen was successfully isolated from the infected root tip tissues and confirmed as C. globosum by microscopic observation. No symptom was observed on the control seedlings. The experiment was repeated three times, and the same results were obtained. For molecular characterization, DNA was extracted from a pure culture of strain XJ160930 with a UNlQ-10 Column Fungal Genomic DNA Isolation Kit (Shanghai Sangon Biotech, China). A 555-bp segment of 5.8S nuclear ribosomal gene with two flanking internal transcribed spacers (ITS), a 676-bp segment of DNA dependent RNA polymerase subunit II gene region (RPB2), and a 733-bp segment of β-tubulin (TUB2) were amplified and then sequenced, using the primers ITS5/ITS4, RPB2AM-1bf/RPB2AM-7R (Miller and Huhndorf 2005), and T1 (O’Donnell and Cigelnik 1997) and TUB4Rd (Groenewald et al. 2013). The sequences, deposited in GenBank as accession numbers KY355130 (ITS), KY355133 (RPB2), and KY355135 (TUB2), showed 100, 99, and 100% sequence identity to C. globosum (GenBank HQ529775.1, KM655411.1, and KX976958.1, respectively). Therefore, both molecular and morphological analyses indicated that the pathogenic fungus was C. globosum. C. globosum has been reported to cause leaf blight disease of Chrysanthemum morifolium in Zhejiang, China (Ma et al. 2015), and decay the stem tissues of the H. brasiliensis (Wong 1988). Here, C. globosum caused failed development of the main roots in rubber seedlings, resulting in susceptibility to wind and proneness to toppling later in their lives. As far as we know, this is the first report of this pathogen involved in disease in H. brasiliensis in China occurring in the early phases of the host life.