Fusarium wilt of Coleus forskohlii caused by Fusarium oxysporum in China

Abstract

Since 2005, a wilt disease on Coleus forskohlii of high severity has been observed in Tongcheng County, Hubei province, China, with the annual crop area affected estimated to be over 100 ha. Symptoms of disease, which included leaves wilting, stem brown discolouration and root rot, were observed after seed germination of C. forskohlii during the spring and summer growing seasons. After isolation and pathogenicity testing, the pathogen was identified as Fusarium oxysporum on the basis of morphological and cultural characteristics. Subsequent analysis of the internal transcribed spacer region of ribosomal DNA and the β-tubulin gene sequence confirmed F. oxysporum as the causal agent. This is the first report of F. oxysporum infecting C. forskohlii worldwide.

Résumé

Depuis 2005, une importante flétrissure a été observée sur Coleus forskohlii dans le comté de Tongcheng de la province de Hubei, en Chine, où la surface en culture touchée est évaluée à plus de 100 ha. Les symptômes de la maladie, qui incluent la flétrissure des feuilles, un changement de coloration de la tige (brun) et le pourridié, ont été observés après la germination de C. forskohlii au cours des périodes de croissance du printemps et de l'été. Après isolation et avoir effectué un test de pathogénicité, Fusarium oxysporum a été identifié, sur la base de ses caractéristiques morphologiques et culturales, comme étant l'agent pathogène. Une analyse plus poussée de la région de l'ITS de l'ARNr et de la séquence du gène β-tubuline a confirmé l'identité de l'agent causal. Il s'agit de la première mention de F. oxysporum infectant C. forskohlii.

Keywords:

• Coleus forskohlii
• Fusarium oxysporum
• outbreak
• root rot
• wilt disease

Mots clés:

• Coleus forskohlii
• éclosion
• flétrissure
• Fusarium oxysporum
• pourridié

Introduction

Coleus forskohlii Briq. is a perennial herbaceous plant in the family Laminaceae, native to India, Nepal, Burma, Sri Lanka, Thailand and China, where it usually grows wild on barren soil and hilly areas. It can also be found growing in Egypt, Arabia, Ethiopia, tropical East Africa and Brazil (Zhao & Jin, 1996; Kavitha et al., 2010), and is widely used for various ailments. For example, in Egypt and other African countries, the leaf is used as an expectorant, emmenagogue and diuretic. In Brazil, it is processed as a stomach aid and for treating intestinal disorders. It is used as a condiment in India, and the tubers are pickled for consumption (Kavitha et al., 2010), and the plant has also been commonly used as a Chinese herbal medicine since ancient times.

In China, C. forskohlii has been artificially cultivated in Yunnan province since 1989, and by 1999, was introduced into central China, where leaves, stems and roots are typically harvested in the autumn. During the winter months, fields are left fallow, and seeding occurs in the spring after deep tillage. A wilt disease was first observed by growers in Tongcheng County, Hubei province in spring 2005, and has been found annually since then. The symptoms of this disease included leaves wilting, stem discolouration and root rot and were first observed soon after seed germination, with the crop area affected estimated to be over 100 ha per year in this single county alone. Due to this disease, yield has been reduced by 20% on average, with up to 100% yield losses in some fields during the spring and summer growing seasons. Initial symptoms are observed on leaves that become stunted, followed by wilting, drooping and dark brown vascular discolouration of stems (Figs 1a , b ). The roots turn brown and become rotted, and the above-ground parts die back.

Fig. 1. a, Fusarium wilt of 5-month-old Coleus forskohlii plants caused by Fusarium oxysporum in a field in Tongcheng County, Hubei Province, China, taken in August 2008. b, Typical symptom of fusarium wilt disease on internal stems of C. forskohlii showing dark brown vascular discolouration.

The objectives of this study were to investigate this new wilt disease of C. forskohlii in central China, and to determine the causal agent through morphological and molecular identification as well as by pathogenicity tests on healthy potted C. forskohlii plants in the laboratory.

Materials and methods

Isolation of the causal agent

In August 2008, diseased plants with typical wilt symptoms were collected from two fields in Tongcheng County which is located approximately 200 km south of Wuhan, on the Hunan Province border. These fields are at 120–180 m elevation in a mountainous region 50 km southeast of the Yangtze River which carves a channel through this region. Small sections (2 × 2 mm) of infected basal stem were surface-sterilized by soaking stem pieces in 70% ethanol and 0.1% mercuric chloride solution for 1 min, rinsed in autoclaved water and placed onto potato dextrose agar (PDA, 2% dextrose, 1.2% agar). The agar plates were incubated at 25 °C on a 12 h light-dark cycle. Cultures were obtained by transferring single conidia onto 2% water agar. All isolates were stored at 4 °C on PDA slants.

Morphological observation of fungus

To identify the fungus isolated from diseased tissues of C. forskohlii, single-conidial isolates were first characterized based on morphological examination. Cultures were grown on PDA in a chamber at 25 °C with a 12-h photoperiod to examine characteristics of colony and colour changes in the medium. Colony diameters were measured after 5, 6 and 7 d. The mean linear growth rate was calculated from sets of five plates, and determined by subtracting the colony diameter on day 5 from the colony diameter on day 7, and divided by 2 for a daily growth rate. After incubation on carnation leaf agar (CLA) for 10 days, macroconidia, microconidia, phialides and chlamydospores of the isolates were examined by microscopy, and compared with previous descriptions (Booth, 1971; Nelson et al., 1983).

Analysis of fungal rDNA-ITS and β-tubulin gene sequence

For DNA sequence comparisons, genomic DNA was extracted from one representative isolate by a CTAB method (Taylor et al., 1993), and fungal rDNA from the internal transcribed spacer (ITS) region and the gene encoding β-tubulin was amplified with primer sets ITS1/ITS4 (White et al., 1990) and T1/T2 (O'Donnell & Cigelnik, 1997), respectively. DNA amplification was performed in a 50 μL reaction mixture containing 5 μL 10 × PCR buffer, 5 μL 25 mM MgCl₂, 2 μL 2.5 mM dNTP, 0.3 μL 5U mL⁻¹ Taq DNA polymerase (Takara, Dalian, China), 1 μL 25 mM of each primer (ITS1 and ITS4, or T1 and T2), 10 ng DNA template and 44.6 μL sterile-ddH₂O. Amplifications were performed with a PTC-100 thermal cycler (Bio-RAD, USA) using the following program: an initial denaturation step at 94 °C for 4 min, followed by 36 cycles of 94 °C for 1 min, 54 °C for 1 min, 72 °C for 1 min and a final extension step at 72 °C for 10 min. Successful PCR reactions resulted in a single band as observed on a 1% agarose gel. PCR products were sent to Genecore (Shanghai) for purification and sequencing. Resulting ITS and β-tubulin sequences were compared with the GenBank database using BLASTN, and the highest scoring matches were downloaded for closer comparison and alignment using DNAMAN (Version 5.2.2) (Perrone et al., 2004).

Pathogenicity tests

Pathogenicity tests were performed in the laboratory on one-month-old 10–15 cm-tall plants of C. forskohlii based on the method of Sharma et al. (2005), with two repeated experiments. There were 20 plants treated for each of two isolates. Seeds of C. forskohlii, provided by Hubei Furen Pharmaceutical Company, were surface-sterilized with 0.5% carbendazim for 30 min before planting in 15-cm-diameter plastic pots containing sand and soil (2 : 1) which had been autoclaved at 121 °C for 2 h. The conidia were produced on PDA at 25 °C under dark conditions for 7 days. Spore suspensions were prepared by first adding autoclaved water to each PDA plate, dislodging spores from the mycelia using a sterile bent glass rod, and then filtering the resulting suspension through three layers of sterile cheesecloth to remove mycelial fragments. The conidial concentration was adjusted to 1 × 10⁵ conidia mL⁻¹. Plants were removed from the pots and roots were washed. Fine root tips of plants (up to 0.5 cm long and 2–5 mm in diameter) were trimmed off and submerged for 5 min in a conidial suspension of each isolate, while wounded roots of control plants were dipped in sterile water. Seedlings were potted in autoclaved soil again, watered every three days to keep soil at a water content of 40–60% and incubated at 25 °C on a 12 h light-dark cycle. Foliar symptoms were assessed daily until disease was observed, and then stem and root symptoms were also observed weekly for up to one month. At the end of this period, isolations were made from diseased tissues, and the pathogen compared with the original inoculated isolates.

Results and discussion

Forty-seven isolates with cultural characteristics similar to Fusarium spp. were obtained from diseased samples. Among isolations from diseased basal stem, 94% were fusarioid species, while the rest of the isolates were identified as species of Alternaria.

One Fusarium isolate from each of two fields were selected to evaluate cultural and morphological characteristics of the pathogen (TC-F-1 and TC-F-10). Morphological characteristics of colony, colouration of medium, macroconidia, microconidia, phialides and chlamydospores of these two isolates were similar. Initial colonies of isolates on PDA were composed of dense, cottony and white mycelium when incubated at 25 °C in 12 h/12 h dark/light photoperiod, but after 5 days on PDA, the colonies developed a dark purple pigmentation on the top surface. Mean linear growth rates at 25 °C for these two isolates were 13.5 mm day⁻¹ and 14.0 mm day⁻¹. After incubation on CLA for 10 days, microconidia of the isolates were abundant, generally unicellular, oval to reniform, 5.5–7.5 × 2.3–5.0 μm, and formed in false heads on short monophialides (5.0–12.8 × 2.1–3.3 μm). Macroconidia were also abundant, mildly sickle-shaped with attenuated apical cells, foot-shaped basal cells, thin-walled and 22.5–50.5 ×3.0–5.0 μm with 3 to 5 septa (mostly 3-septate). Chlamydospores were abundant, globose, single-celled, terminal or intercalary, produced singly or in pairs and 7.0–10.0 μm in diameter. Based on cultural characteristics and fungal morphology on PDA and CLA, the isolates TC-F-1 and TC-F-10 were identified as F. oxysporum.

Dimensions and morphology of microconidia, macroconidia and chlamydospores as well as descriptions of conidiogenesis were similar to those reported by Booth (1971) and Nelson et al. (1983). The major morphological characteristics used in the description of Fusarium species include macroconidial and microconidia morphology and conidiogenesis (Summerell et al., 2003). Although with seemingly uniform morphological features, F. oxysporum is genetically diverse, and pathogenic strains have been assigned to formae speciales based on host specificity, with over 120 formae speciales currently described (Michielse & Rep, 2009). Further work is needed to determine the formae specialis of the pathogen by combination of host range tests and molecular markers.

Genomic DNA (approximately 50 ng μL⁻¹) was extracted from isolate TC-F-1, and successful PCR reactions with the ITS and β-tubulin primer sets both resulted in a single band. In early 2012, comparison of the 505-bp ITS sequence (GenBank Accession No. JQ950332) with GenBank nr database showed 100% identity with six accessions of F. oxysporum and 18 accessions of Gibberella moniliformis (sexual state of F. verticillioides) as the top matches. Moreover, comparison of the 412-bp β-tubulin sequence (GenBank Accession No. JQ950333) with Genbank nr showed 99.6% identity with β-tubulin in seven accessions of F. oxysporum and one accession of F. solani as the top matches. According to the results of sequence comparisons, two other species, F. verticillioides and F. solani, also had highest identity with the tested isolate using ITS or β-tubulin sequence data. However, Nelson et al. (1983) reported that the distinguishing characteristics are microconidia forming in chains on monophialides and the absence of chlamydospores for F. verticillioides (syn: F. moniliforme), and the large macroconidia, elongate monophialides bearing microconidia, and the distinctive cream, blue-green or blue colour of colonies on PDA for F. solani. The putative F. oxysporum isolates found in this study did not share these characteristics. Thus, identification of the causal agent of fusarium wilt of C. forskohlii in China as F. oxysporum is supported by morphological characteristics and DNA sequence data. The fungal isolate TC-F-1 has been deposited in the China Center for Type Culture Collection (CCTCC), with the accession number CCAM041406.

All C. forskohlii plants developed disease symptoms after inoculation with conidial suspensions of the two isolates. The leaves became wilted within 14 days after inoculation, inner stems turned dark brown, and brown rotted roots were observed. Plants started to die three to four weeks after inoculation. Symptoms were similar to those observed on infected plants in the field. None of the controls treated with sterile water developed symptoms. Koch's postulates were fulfilled by re-isolating from the surface-sterilized stem of inoculated plants, and recovering colonies with the same morphological features as these originally isolated. Results of the two repeated experiments were similar.

The moderate temperature and precipitation in Tongcheng County where C. forskohlii is grown may be favourable to the development of this fusarium wilt disease. During the spring and summer growing seasons, in one plantation under study which was located on a hillside, the mean daytime temperature range during the last three years was 22–25 °C, and monthly precipitation was 100–190 mm. However, we speculated that continuous cropping was the most important factor for the outbreaks of fusarium wilt of C. forskohlii. In Tongcheng County, local farmers had been planting only C. forskohlii every year for its high market value since 1999, and this was likely highly conducive to the build-up of F. oxysporum inoculum in the fields.

A survey of the literature revealed only a few fungal diseases associated with C. forskohlii. These include wilt disease caused by F. chlamydosporum or F. solani (Shyla, 1998; Anirban & Sabita, 2008), root rot caused by Macrophomina phaseolina (Kamalakannan et al., 2005), leaf spot caused by Botryodiplodia theobromae (Ramprasad, 2005), leaf blight caused by Rhizoctonia solani and stem blight caused by Phytophthora nicotianae var. nicotianae (Shukla et al., 1993; Singh et al., 2011), which all were found in India, in addition to a report of leaf spot disease caused by Corynespora cassiicola in Brazil (Fernandes & Barreto, 2003). Moreover, although F. oxysporum is a cosmopolitan plant pathogen, it has not been reported on any species in the genus Coleus. To our knowledge, this is the first report of F. oxysporum infecting C. forskohlii worldwide.

Acknowledgements

This work was supported by National Key Technology R&D Program in the Eleventh Five Year of China (No. 2006BA09B04-06).