A strain of Coprinellus curtus (designated GM-21), a basidiomycete that suppressed bottom-rot disease of Chinese cabbage, ‘pak-choi’ (Brassica campestris), caused by the pathogen Rhizoctonia solani Pak-choi 2 was isolated. The mechanism of plant disease suppression was discovered to be hyphal interference, a combative fungal interaction between strain GM-21 and the pathogen. The antifungal spectrum of strain GM-21 was shown to include R. solani and Fusarium sp., i.e. strain GM-21 showed disease-suppressive ability against bottom-rot disease of lettuce and Rhizoctonia-patch disease of mascarene grass caused by strains of R. solani. In addition, clear evidence of hyphal interference between strain GM-21 and Fusarium pathogens that cause crown (foot) and root-rot disease of tomato and Fusarium wilt of melon, respectively, was demonstrated. It was thus considered that GM-21 is effective for suppressing soil-borne pathogens, and that GM-21 presents new possibilities for biological control of vegetable diseases.
In order to suppress vegetable diseases without the use of (or with reduced use of) chemical pesticides, a great deal of research has been carried out on the use of biological control (e.g. Kumari & Srivastava, 1999; Dube, 2001; Punja & Utkhede, 2003; Roberts & Lohrke, 2003; Haas & Defago, 2005). Biological control of soil-borne plant pathogenic fungi depends on various mechanisms, which include: the production of antibiotics, lytic enzymes or secondary metabolites (Bruce et al., 1995; Thomashow et al., 2002; Yu et al., 2002; Liu & Li, 2005; Loper et al., 2007); competition for nutrients (Janisiewicz et al., 2000); hypovirulence (Milgroom & Cortesi, 2004); competitive exclusion principles (Bacon et al., 2001); and mycoparasitism (De Vrije et al., 2001). Furthermore, a systemic resistance in the plant against infection by fungal pathogens can be introduced by both pathogens and nonpathogenic rhizobacteria (van Loon et al., 1998; Khan et al., 2006).
Pak-choi (Brassica campestris) is one of the most important vegetables grown in the countryside near Hamamatsu city, Japan. Suppression of bottom-rot disease caused by Rhizoctonia solani that afflicts pak-choi has been necessary for many years. We found healthy pak-choi grown in certain areas outside but adjoining the affected area. The aims of the present investigation were (1) to find a microorganism that will help to suppress bottom-rot disease and that therefore can be used in biological control of this plant disease, and (2) to investigate the mechanisms of the suppression of the disease.
Materials and methods
Plant and pathogen
Pak-choi (B. campestris L. chinensis group) was used for a plant disease suppression test. Plant seeds were first surface sterilized with 1% benzalkonium chloride (v/v) for 6 s, then with 70% ethanol (v/v) for 30 s, and then finally with 1% sodium hypochlorite (v/v) for 20 min, successively. After surface sterilization, seeds were rinsed thoroughly with sterilized water. The pathogen used was R. solani Pak-choi 2 provided by the Shizuoka Agricultural Experiment Station, which causes bottom-rot disease of pak-choi. The pathogen was maintained on water agar at 4 °C.
Soil was collected from farmland with gray lowland soil, where pak-choi grew without disease symptoms. The pak-choi was grown in the farmland for over 5 years with application of both organic and chemical fertilizers. The physicochemical properties of the soil were as follows: pH (H2O) 6.41, EC 0.35 mS cm−1, organic matter 3.11%, P2O5 2.32 mg g−1, K2O 0.16 mg g−1, MgO 0.92 mg g−1, CaO 4.71 mg g−1. Soil samples were collected from the upper layer (0–10 cm depth) of the soil. The samples were immediately used for isolation of microorganisms.
Isolation of suppressive fungi from the soil
Fungi were isolated from the soil by the dilution plating method using potato dextrose agar (PDA; Eiken Chemical Co., Ltd, Tokyo) supplemented with 30 µg L−1 streptomycin. Three grams (wet weight) of a soil sample was suspended in 27 mL of sterile water in a sterilized homogenizer cup and dispersed at 10 000 r.p.m. for 10 min with a homogenizer (Model EX-3, Nihon Seiki Ltd, Tokyo). After serial dilution, 100 µL of suspension was spread on the agar plates, and the plates were then incubated at 27 °C for 7 days. Isolates were transferred to a water agar slant and were maintained at 4 °C.
Disease suppression assay using the isolated fungi
Eight isolated fungi were examined for their abilities to suppress bottom-rot disease by using the disease suppression assay. Nine potting mixes were prepared including the eight isolated fungi, plus one control. The soil was sterilized by autoclaving at 121 °C for 90 min to delete the effect of microorganisms indigenous to the soil. Each fungus was precultured in potato dextrose (PD) liquid medium at 27 °C for 7 days, and then the culture broth was filtered with six layers of sterile cotton gauze. The fungal mycelium collected on the gauze was suspended in the sterilized water, and a mycelial suspension of each fungus with a concentration of approximately 0.6 g (wet wt)L−1 was prepared.
The pathogen, R. solani Pak-choi 2, was precultured on 9-cm Petri plates containing PDA at 27 °C for 7 days, and approximately 5 × 5 cm of the agar plate together with R. solani mycelium was then cut out and homogenized in sterilized water at 10 000 r.p.m. for 10 min. It was then diluted tenfold to eliminate agar debris and the pathogen suspension was prepared at a density of 0.4 g (wet wt)L−1.
At the start of the experiment, 1 mL of each fungus suspension and 2 mL of the pathogen suspension were inoculated into 50 g of the sterilized soil. Sterilized water was then added to all mixes during hand blending with a sterilized spatula in order to bring the moisture level to 50% (w/w).
The mixes (approximately 60 g wet wt) were then distributed into polycarbonate pots (70 mm in diameter, 121 mm in height; AGRIPOT-1, Asahi Techno Glass Corp., Funabashi, Japan), and 20 surface-sterilized pak-choi seeds were planted in each pot. Aseptic conditions were maintained throughout the preparation and the experimental periods. The experiment was conducted for 32 days. In order to evaluate suppressive ability, three pots were prepared for each mix. The pots were placed randomly in a growth chamber (BEC-II-350HUP; Shimadzu Rika Corp., Tokyo) and incubated. The humidity in the growth chamber was controlled at 50% throughout the experiment, and the temperature and illumination were set as follows: (1) increasing temperature from 20 to 30 °C at 5000 lux (00 : 00 to 04 : 00 h), (2) constant temperature at 30 °C at 10 000 lux (04 : 00 to 12 : 00 h), (3) decreasing temperature from 30 to 20 °C at 5000 lux (12 : 00 to 16 : 00 h), and (4) constant temperature of 20 °C under darkness (16 : 00 to 00 : 00 h). Disease severity was rated by scoring the disease development on a scale from 0 to 5, where 0 was asymptomatic pak-choi, 1 was 1–20% lesion development, 2 was 21–40% lesion development, 3 was 41–60% lesion development, 4 was 61–80% lesion development, and 5 represented 81–100% lesion development.
The disease severity for one potting mix was obtained by averaging the values of disease severity for three pots. Disease severity data were then subjected to a one-way analysis of variance (anova) with Tukey's multiple range tests for the mean values measured at 0, 11, 21 and 32 days after the start of the experiment.
Observation of mycelial interaction between strain GM-21 and the pathogen
A paired culture of two strains, GM-21 and R. solani Pak-choi 2, was prepared as follows. The two fungi were placed on opposite sides of a 9-cm Petri dish containing PDA and then incubated at 27 °C until the two mycelia made contact. For microscopic observation, a sterilized rectangular piece of cellophane was placed between the two fungi at the beginning of inoculation (Ikediugwu & Webster, 1970). When the mycelia of the two strains made contact on the cellophane slip, the slip was peeled off. Mycelia of the two strains on the cellophane slip were stained with 0.25% (w/w) lactophenol-cotton blue for 10 s.
The antifungal spectrum of strain GM-21
In order to investigate the antifungal spectrum of strain GM-21, a disease suppression assay of two herbaceous plant diseases was carried out. One involved bottom-rot disease of lettuce caused by R. solani lettuce 2, and the other involved Rhizoctonia-patch disease of mascarene grass (Zoysia tenuifola Willd.) caused by R. solani Kuhn AG2-2 (Nakasaki et al., 1998). Each pathogen was provided by the Shizuoka Agricultural Experiment Station and was isolated by our laboratory. The experimental methods were the same as those used for the pak-choi disease.
In addition to the disease suppression assay, using the same method mentioned above for strains GM-21 and Pak-choi 2, we conducted a paired culture of strain GM-21 and two Fusarium strains, i.e. Fusarium oxysporum f.sp. radicis-lycopersici and Fusarium oxysporum f.sp. melonis. These pathogens (also provided by the Shizuoka Agricultural Experiment Station) cause crown (foot) and root-rot disease of tomato and Fusarium wilt of melon, respectively.
Results and discussion
Eight fungal isolates, A–H, were isolated from a suppressive soil. These isolates were examined for their abilities to suppress bottom-rot disease of pak-choi by means of a disease suppression assay. In control experiments, the pak-choi showed signs of bottom-rot disease immediately after the start of experiment, and the disease incidence rapidly increased to 100% (Table 1). In contrast, when isolate G was used, disease incidence was maintained at 0% throughout the full duration of the experiment. Disease incidence for the other isolates reached almost 100%, except for the case of isolate D, though the time course was dependent on the isolate used. Isolate G, which had an extremely high suppression ability, was designated strain GM-21 and was selected for further investigation. Although not shown here in detail, we isolated 12 bacterial strains, including actinomycetes that were prevalent in the soil, by a dilution plating method using trypticase soy agar medium; none showed suppressive effects under the present experimental conditions.
|Disease severity (%)|
|Cultivation time (days)|
|Disease severity (%)|
|Cultivation time (days)|
Isolate G was identified as Coprinellus curtus GM-21.
Different letters represent a significant difference as determined by Tukey's multiple range test (P<0.05).
Strain GM-21 grew on sterilized soil, and formed a fruiting body (Fig. 1) as well as basidiospores on the basidium after 7 days of cultivation at 27 °C. In addition to these observations, similarity analysis of internal transcribed spacer (ITS) — 5.8S rRNA gene sequences (DDBJ accession no. AB266447) amplified by using the following set of PCR primers: forward, 5′-GGAAGTAAAAGTCGTAACAAGG-3′, and reverse, 5′-TCCTCCGCTTATTGATATGC-3′(White et al., 1990), identified strain GM-21 as representing Coprinellus curtus (Fig. 2; 99.8% sequence similarity).
A photograph of the paired culture of the two strains, GM-21 and Pak-choi 2, on opposite sides of the PDA plate is shown in Fig. 3. Each strain grew vigorously, and the hyphae of GM-21 interfered with those of the pathogen. At the boundary between the two growth areas, a ‘stalemate’ occurred, in which neither mycelium could enter the area of the other, similar to the description of the mycelial interaction between two wood-decaying fungi given by Rayner & Boddy (1988).
In addition, microscopic observations showed that the hyphae of the pathogen were damaged at the boundary. Figure 4 shows micrographs of the pathogen: the healthy hyphae in the pure culture (Fig. 4a) and the damaged hyphae during confrontation with C. curtus GM-21 in the paired culture (Fig. 4b). The affected cells of the pathogen were stained lightly or not at all with lactophenol-cotton blue, in contrast to the unaffected cells, which stained strongly. Ikediugwu & Webster (1970) suggested that the inability to stain deeply with cotton blue for this kind of damaged hyphae may be due not only to a loss of cell contents but also to chemical alterations to the cytoplasmic constituents. In addition, Fig. 4b shows that the affected hyphae had a granular cytoplasm and were vacuolized. These microscopic observations are consistent with the characteristics of hyphal interference shown in previous research (Ikediugwu & Webster, 1970; Traquair & Mckeen, 1977; Deacon, 1983). From the above results, we ascertained that strain GM-21 suppresses the pathogenic fungi via hyphal interference.
We then ascertained the width of the antifungal spectrum of strain GM-21 by conducting assays for bottom-rot disease in lettuce caused by R. solani lettuce 2 and for the Rhizoctonia-patch disease in mascarene grass caused by R. solani Kuhn AG2-2. Disease incidences for both the lettuce and the mascarene grass increased rapidly to 100% after the start of the experiment without inoculation by strain GM-21 (the control). By contrast, disease incidence remained at almost zero throughout the experiment when the soil containing lettuce and mascarene grass were incorporated with strain GM-21 (data not shown). Moreover, clear hyphal interference was observed between strain GM-21 and the two Fusarium strains, i.e. F. oxysporum f. sp. radicis-lycopersici FO-To-3, and F. oxysporum f. sp. melonis FO-Me-2. These results suggest that C. curtus GM-21 antagonizes another important plant pathogen.
Interestingly, the first description of hyphal interference, to our knowledge, was made with respect to the relationship between Coprinus heptemerus [a fungus related to GM-21; Coprinus being a synonym for Coprinellus (Kirk et al., 2001)] and coprophilous fungi, namely Pilobolus crystallinus and Ascobolus crenulatus (Ikediugwu & Webster, 1970). The purpose of the previous research, however, was to investigate the relationship between C. heptemerus and the coprophilous fungi, and the authors did not mention the suppressive effects on pathogenic fungi.
Hyphal interference between wood decay basidiomycetes and fungi with the ability to suppress them has attracted interest, and attempts have been made to use it as a biological control to prevent wood decay (Ikediugwu et al., 1970; Deacon, 1983; Rayner & Boddy, 1988; Boddy, 2000; Cox & Scherm, 2006). There have been some successful applications; for example, the use of Peniophora gigantea to control the pathogen Heterobasidion annosum in forestry plantations was successful (Ikediugwu et al., 1970), as was the use of Fomes annosus to control wood stump decay, although the term ‘hyphal interference’ was not used to explain this suppression effect (Rishbeth, 1952).
There have been a few examples of biological control for herbaceous plants that depended on hyphal interference; Upadhyay & Bharat Rai (1987) investigated the antagonism between Fusarium udum Butler, which causes wilt of the pigeon-pea, and the saprophytic microflora of the root region of the host plant with reference to colony interaction, and found out that some species of fungi in the genera Aspergillus and Penicillium were effective antagonists against F. udum, although a disease-suppressive assay using pigeon-pea was not carried out. The authors reported that Coprinus lagopus Fr., which is a relative of the fungus used herein, did not show any effect of hyphal interference against Fusarium. Perello (2002) investigated the interaction between foliar pathogens and the saprophytic microflora of wheat phylloplane, and observed that Paecilomyces lilacinus showed the greatest capacity for hyphal interference against the pathogens. Furthermore, they saw the possibilities of this mechanism of hyphal interference for biological control.
As mentioned above, there have been several reports regarding the biological control of disease via mechanisms of hyphal interference; however, the current study provides the first report on the suppression of vegetable diseases caused by soil-borne pathogens by means of hyphal interference with C. curtus, a fungus that has only recently been described. However, this study provides the first report on the suppression of vegetable diseases caused by soil-borne pathogens by means of hyphal interference with C. curtus, a fungus that has never been described. The absence of such reports may be due to the fact that the above-ground part, the fruiting body, of the suppressive fungi tends to disappear immediately after its formation from the soil surface, as with that for C. curtus GM-21 isolated in the present study. Therefore, the existence of such suppressive fungi in farmland may go unnoticed. In fact, this characteristic is clearly expressed in the Japanese name for this fungus, ‘Hitoyo-take’: ‘Hitoyo’ for overnight, and ‘take’ for mushroom.
Coprinellus curtus GM-21 significantly reduced the severity of diseases caused by soil-borne pathogens for herbaceous plants, including vegetables. Microscopic observations of two fungal interactions indicated the importance of hyphal interference against the pathogens in the fungi's antagonistic activity. We consider that the results of the present study provide important evidence that biological control approaches using the mechanism of hyphal interference represent a promising step towards elaborating biological pest management programs for edible herbaceous plants such as vegetables and fruits.
We would like to express sincere thanks to Mr Masayuki Togawa, researcher of the Shizuoka Agricultural Experiment Station, who kindly provided the pathogenic fungi, and gave us critical comments. This research was supported partly by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan.