Identification of Phoma macdonaldii Boerema causing black stem of sunflower in China

Abstract

A severe disease was observed on sunflower (Helianthus annuus L.) in Ili, Xinjiang China, in 2005. The causal agent was isolated from infected tissues, and its pathogenicity confirmed. Pathogenicity tests showed that the fungus could infect sunflower, which developed the same symptoms under artificial inoculation conditions to those observed in the field. The fungus was identified based on morphological and cultural characteristics as Phoma macdonaldii Boerema. Identifications were confirmed by comparison of the 18S rDNA partial sequence, 28S rDNA partial sequence, and the internal transcribed spacer (ITS) regions 1 and 2, including 5.8S rDNA (ITS1‐5.8S-ITS2). This is the first report of black stem of sunflower caused by P. macdonaldii in China.

Résumé

En 2005, une grave maladie a été observée sur le tournesol (Helianthus annuus) à Ili, dans la province du Xinjiang en Chine. L'agent causal a été isolé à partir de tissus infectés et sa pathogénicité, confirmée. Les tests de pathogénicité ont montré que le champignon pouvait infecter le tournesol qui développait les mêmes symptômes après inoculation dans des conditions artificielles que ceux observés au champ. Le champignon a été identifié en tant que Phoma macdonaldii Boerema, et ce, en fonction de ses caractéristiques morphologiques et culturales. L'identification a été confirmée en comparant les séquences partielles 18S et 28S de l'ADNr ainsi que les régions 1 et 2 de l'espaceur transcrit interne, y compris 5.8S de l'ADNr (ITS1‐5.8S-ITS2). Il s'agit de la première mention de la maladie des taches noires sur le tournesol en Chine.

Key words:

• black stem
• Helianthus annuus
• Phoma macdonaldii

Mots clés:

• Helianthus annuus
• maladie des taches noires
• Phoma macdonaldii

Introduction

Black stem is a destructive disease of sunflower (Helianthus annuus L.), and occurs in some countries in the Americas, Europe, Asia and Oceania (McDonald, 1964; Donald et al., 1986; Miric et al., 1999; Debaeke & Peres, 2003; Boerema et al., 2004) and can cause 10–30% yield loss (Larfeil et al., 2002).

Sunflower is one of the most important oil crops grown widely in the north of China, including Jilin, Inner Mongolia, Ningxia, Gansu, Xinjiang, Hebei, Shanxi, and Heilongjiang provinces. In 2005, black stem was found in imported seed-growing fields of sunflower in Ili, Xinjiang, China. In 2006 the disease was more severe and caused an epidemic in Ili province. Diseased sunflower plants were collected and the pathogen was observed microscopically. Fungal morphology was similar to Phoma macdonaldii (Zhao Zhenyu, personal communication). Since then, black stem has also been found in some fields planted to imported sunflower seeds in different provinces. The aim of this study was to determine the pathogen causing sunflower black stem in Xinjiang, China.

Materials and methods

Isolation and characterization of the pathogen

Stems and petioles showing typical black lesions (Fig. 1 a–c) were collected from seven sunflower fields in Ili, Xinjiang, China in 2010. Diseased stem tissues were cut into 2–3 mm pieces, and sterilized with 10% NaOCl for 2 min. The samples were washed three times with sterile distilled water, and plated on potato dextrose agar (PDA) and incubated at 25 °C in darkness for 7 days. Isolates were purified by single spore culturing according to Roustaee et al. (2000) and subcultured on oatmeal agar (OA) at 25 °C with a 12 h photoperiod, in order to characterize fungal growth and morphology.

Fig. 1. Symptoms of black stem of sunflower caused by natural infections in the field and greenhouse inoculations with Phoma macdonaldii. (a) Natural infections on stems of field-grown plants. (b) Symptoms of sunflower black stem on cotyledon petioles in field-grown plants. (c) Pycnidia formed on stems of naturally infected sunflowers in the field. (d) Chlorotic lesions on the stem surface 1–2 days after artificial inoculation. (e) Black lesions on the surface of the cotyledon petiole and stems 7 days after inoculation. (f) Development of pycnidia on oat agar culture. (g) Conidia on OA culture. Bars: a–b, e = 5 cm; d = 1 cm; f = 100 μm and g = 10 μm.

Pathogenicity test

Thirty-five single spore isolates from 11 field isolates were tested for pathogenicity on sunflower. Twenty μL spore suspensions containing 1 × 10⁶ spores mL⁻¹ were inoculated onto the cotyledon petiole and hypocotyls of 2-week-old sunflower seedlings (10 seedlings per isolate) using a micropipette technique (Roustaee et al., 2000). The inoculated seedlings were incubated at 25 °C (day)/18 °C (night), with a 14 h photoperiod, and a relative humidity of 75–80% in a growth chamber. The test was repeated twice for each isolate.

Molecular identification

For phylogenetic analysis, genomic DNA of 11 isolates collected in Ili, Xinjiang was extracted using a QIA quick gel extraction kit (Qiangen, Hilden, Germany) and the 18S rDNA, 28S rDNA and ITS1‐5.8S-ITS2 regions were amplified with primers ITS1 and ITS4 (White et al., 1990) under the following reaction conditions: 94 °C for 5 min, 35 cycles of 94 °C for 30 s, 56 °C for 30 s, 72 °C for 45 s and a cycle of 72 °C for 10 min. The amplified products were resolved by electrophoresis in a 1.2% agarose gel.

Results and discussion

Isolation and characterization of the pathogen

The colonies on OA were greyish-white with black particles, with pure white aerial mycelium with a woolly-cottony appearance. Pycnidia (Fig. 1f ) were dark-brown, formed on the aerial mycelium and contained slimy spore masses. Pycnidia (n = 30) were 70–165 μm in diameter, initially appearing in the centre of the colonies, extending to the colony edge after 7 days. Conidia (Fig. 1g ) were hyaline, ellipsoid or cylindrical, 4.5–10.0 × 2.0–3.5 μm in diameter. The morphology of isolates was similar to that described for Phoma macdonaldii (Boerema et al., 2004).

Pathogenicity test

Symptoms of black stem were observed in all of the inoculated sunflower seedlings, but not in the non-inoculated control seedlings. Small chlorotic lesions (Fig. 1d ) appeared on the surface of the cotyledon petiole 1–2 days after inoculation and spread up and down the stems 7 days later (Fig. 1e ). The fungus was re-isolated from all inoculated seedlings for each isolate and was identical to the original isolate. The symptoms were similar to those observed in the field (Fig. 1a –c) and that described for sunflower black stem (Roustaee et al., 2000; Larfeil et al. 2002).

Molecular identification

The target band, with a predicted size of 583 bp (Fig. 2), was retrieved with DNA Fragment Quick Punifiction Kit (Dingguo, Changsheng, China) and sequenced (Shanghai Integrated Biotech Solutions Co., Ltd) and used to search the nucleotide database of GenBank using the Blastn. The searches revealed that eight sequences of 11 isolates shared 100% identity each other, and 99% identity with the other three isolates, and all of the sequences shared 99% similarity with P. macdonaldii (Accession No. HM003262) and 88–92% similarity with other species of Leptosphaeria (teleomorph of Phoma). The sequence of one representative isolate (Pho1) was deposited in GenBank (Accession No. HQ700313). A homology tree was constructed with ITS nucleotide sequences from 13 isolates representing different Leptosphaeria species (Fig. 3). The multiple sequence alignment was carried out using cluster analysis software (DNAMAN). These results confirmed the identity of the fungus as P. macdonaldii.

Fig. 2. Agarose gel showing amplification of ITS fragments from isolates of Phoma macdonaldii. Lanes 1–11: isolates of P. macdonaldii. Ck: negative control. M: 100bp DNA Ladder Plus (Shanghai Biotechnology CO., LTD. China).

Fig. 3. Homology tree constructed with the nucleotide sequences of the internal transcribed space regions of isolates from 13 different Leptosphaeria species, including one new isolate from this study (HQ700313), and 12 isolates retrieved from GenBank.

Acknowledgements

This work was supported by the National Natural Science Foundation of Peoples' Republic of China (30960217). The authors wish to thank Dr Z. Y. Zhao for identifying the collection of P. macdonaldii.