1. Introduction
The term phylloplane refers to the parts of plants above ground and dominated by leaves and is an important habitat for microorganisms [
1]. Growth of phylloplane microorganisms depends on organic compounds secreted from the plant itself and organic compounds from external sources [
2,
3]. The phylloplane of plants in both temperate and tropical regions has been reported to be colonized by yeasts belonging to both phyla, Ascomycota and Basidiomycota, although the majority of the strains are in Basidiomycota [
4,
5,
6,
7,
8].
Rice is one of the most widely produced and consumed staple foods in the world, especially in Asia [
9]. In Thailand the rice cultivation area in the year 2017/2018 was approximately 59.2 million hectares, with a production of 24.9 million tons (
http://www.oae.go.th/view/1/). The rice species cultivated in Thailand is commonly known as Asian rice (
Oryza sativa L.). One of the major causes of decreases in rice production is diseases caused by pathogenic fungi. The major rice diseases caused by fungal pathogens in Thailand are blast (caused by
P. oryzae), sheath blight (caused by
R. solani), bakanae (caused by
F. moniliforme), brown spot (caused by
H. oryzae) and dirty panicle (caused by
Cu. lunata and
H. oryzae) (Rice Department, Ministry of Agriculture and Cooperatives of Thailand, 2014). Rice sheath blight disease causes yield losses of 25%–35% of Thai rice production [
10]. This disease is the second most important rice disease worldwide [
11].
The management of rice diseases caused by fungi is mainly based on the use of the chemical fungicides such as Carbendazim
®, Validamycin
®, Propiconazole
® and Mancozeb
® [
12,
13,
14]. However, the use of chemical fungicides is not a long-term solution and is becoming less acceptable due to increasing residues, toxicity to non-target organisms and other health and environmental hazards [
15]. Biological control is an environmentally friendly alternative approach to plant disease management. In the last two decades, biological control based on using antagonistic yeast has been demonstrated. Antagonistic yeasts have been sought for use as biological control agents for plant and post-harvest diseases. For example,
Papiliotrema (
Cryptococcus)
flavescens and
Sporobolomyces roseus, reduced lesion density and necrosis of red stalk rot of maize caused by
Colletotrichum graminicola when applied as a mixture to maize plants [
16],
Saccharomyces cerevisiae,
Candida albicans and
Candida sake were reported to significantly reduce the powdery mildew and Cercospora leaf spot diseases on sugar beet [
17] and
Rhodotorula (
Rhodosporidium)
kratochvilovae and
Papiliotrema (
Cryptococcus)
laurentii UM108 suppressed the powdery mildew disease on wheat [
18].
Antagonistic yeasts use both direct and indirect mechanisms to inhibit plant pathogens, including production of volatile organic compounds, cell wall degrading enzymes, siderophores and biofilm as well as competition of nutrients, and phosphate and zinc oxide solubilization [
18,
19,
20,
21,
22,
23,
24,
25]. Volatile organic compounds (VOCs) are substances with a low molecular weight (lower than 300 Da), low polarity and high vapor pressure [
26]. Emission of VOCs by antagonistic yeasts have proven to be one of the important direct antagonistic mechanisms against pathogenic fungi.
Candida intermedia was found to produce VOCs that inhibited mycelium growth of
Botrytis cinerea [
27].
Candida maltosa NP9 emitted VOCs that inhibited spore germination of
Aspergillus brasiliensis [
28].
Sporidiobolus pararoseus was found to produce VOCs that effectively inhibited both the conidial germination and the mycelial growth of
B.
cinerea [
29]. Hua et al. [
30] reported that VOCs produced by
Wickerhamomyces anomalus WRL-076 inhibited growth and aflatoxin production of
Aspergillus flavus. Secretion of fungal cell wall degrading enzymes, especially β- 1, 3-glucanase and chitinase, by antagonistic yeasts is one of the direct antagonistic activities against pathogenic fungi. These enzymes hydrolyze polymeric compounds in the fungal cell wall, and this directly suppresses activities and/or induces death.
Candida oleophila,
Meyerozyma guilliermondii and
Pichia membranifaciens were reported to produce β- 1, 3-glucanase and chitinase, which kill pathogenic fungi [
22].
M. guilliermondii produced β-1,3-glucanases and chitinase, and higher production of both enzymes was found when cultivated in a medium supplemented with cell wall fragments of
Colletotrichum capsici [
31].
P. membranifaciens suppressed the growth of
B. cinerea through the production of β-1,3-glucanases [
32].
Meyerozyma caribbica showed a high antagonistic potential against
Colletotrichum gloeosporioides in mango through the production of hydrolytic enzymes such as chitinase and β-1,3-glucanase [
23]. Siderophores are secondary metabolites which are low molecular weight compounds with a high affinity for iron. When siderophore producing antagonistic microorganisms are applied in the agricultural field, they suppress the pathogens’ growth or reduce their metabolisms by competition for iron, resulting in a decrease in the pathogens [
33].
Rhodotorula glutinis has been shown to produce rhodotorulic acid, a hydroxamate type siderophore, which suppresses various plant pathogenic fungi [
34]. This antagonistic yeast species was also reported to reduce
B. cineria spore germination and disease caused by this pathogenic fungus in biocontrol experiments on apple fruit [
35]. Biofilm is a group of microbial cells embedded within a matrix of extracellular polymeric substance produced by the cells, which adhere to a surface. Biofilm formation on a plant will protect the plant from destruction by pathogens and is related to the competition for nutrients on the surface of the plant [
23,
36]. In addition, yeasts cells in biofilm can destroy fungal pathogens by secretion of fungal cell-wall degrading enzymes during the adhesion process [
36]. Competition for nutrients between antagonists and pathogenic fungi is among the direct antagonistic mechanisms. This mechanism is based on the ability of antagonistic yeasts to rapidly colonize and multiply on the plant surface and subsequently to compete with the pathogens for nutrients and space [
24,
31,
37]. Reduced efficiency of
W. anomalus and
S. cerevisiae against
Pichia digitatum in orange was obtained from this mechanism [
38]. Tian et al. [
39] reported that
Metschnikowia pulcherrima could compete for nutrients under in vivo condition when glucose was added to wounds on mango fruits. Some microorganisms are capable of solubilizing insoluble essential elements such as phosphate and zinc oxide in the environment to their soluble forms, which are subsequently uptaken by plants [
21].
This work aimed to study yeasts in the phylloplane of rice, to evaluate the antagonistic activities and mechanisms against rice pathogenic fungi of the rice phylloplane yeasts and to evaluate the efficacy of the selected antagonistic yeasts for controlling the rice sheath blight disease in rice plants in a greenhouse.
4. Discussion
In this work we isolated rice phylloplane yeasts by plating of leaf washing using YM agar and obtained a higher number of yeast strains in phylum Basidiomycota (84.4%) than in the phylum Ascomycota (15.6%). This result is in accordance with previous reports when the same isolation technique, the plating of leaf washings, was used to isolate yeast from sugarcane phylloplane for a diversity study in Brazil [
4] and in Thailand [
60]. Both investigations reported a majority of basidiomycetous yeast strains and a smaller number of ascomycetous strains. In the present study,
Moesziomyces antarcticus was found to be present in as many as 55 rice leaf samples. Our result agrees well with the result of Nasanit et al. [
7] that
M. antarcticus was the most frequently detected species in the rice phylloplane when a culture-independent method was used to assess yeast diversity. Among 50 species obtained from rice phylloplane, ten species (
C. tropicalis,
D. nepalensis,
M. antarcticus, M. aphidis P. flavescens,
P. laurentii,
P. rajasthanensis,
R. taiwanensis and
Sp. blumeae) were also detected when an enrichment isolation technique was used [
61].
Various yeast species in both phyla have been reported to have antagonistic activity against plant pathogenic fungi. Examples of ascomycetous yeast species were
C. oleophila,
C. sake,
Hanseniaspora uvarum,
K. ohmeri,
Metschnikowia fructicola,
M. guilliermondii,
S. cerevisiae and
Torulaspora globosa, and examples of basidiomycetous yeast specieswere
Cryptococcus albidus,
P. laurentii and
Sp. pararoseus [
21,
25,
62,
63,
64,
65]. In the present study, only 83 yeast strains out of 282 strains with active growing were evaluated for their antagonistic activities. This was because many yeast strains showed very weak growth or lost their viability after preservation at −80 °C for many months. The results revealed that 14 strains of five yeast species were capable of inhibiting growth of one to five rice pathogenic fungi, namely
Cu. lunata,
F. moniliforme,
H. oryzae,
P. oryzae and
R. solani. Among the inhibiting species,
T. indica and
W. anomalus inhibited all five rice pathogenic fungi. The results indicated that among microflora associated with rice phylloplane, some antagonistic yeasts that have potential to control rice diseases caused by fungi are present. In this study, only ascomycetous yeast species were capable of antagonizing these fungal pathogens of rice. Some antagonistic yeast species found in the present study have been reported previously.
M. guilliermondii could control the chilli anthracnose fungus after harvesting [
31].
K. ohmeri revealed growth inhibition of
Penicillium expansum, a postharvest pathogenic fungus [
62].
W. anomalus was reported for its biocontrol activity against
Alternaria alternata,
Aspergillus carbonarius,
Botrytis cinerea,
Monilinia fructicola and
P. digitatum [
66]. Among
Torulaspora species, only
T. globose was previously reported for its biocontrol activity against
Colletotrichum sublineolum in sorghum [
21]. This is the first report on the strong antagonistic activity of
T. indica strains against pathogenic fungi causing rice diseases, namely
Cu. lunata,
F. moniliforme,
H. oryzae,
P. oryzae and
R. solaniDirect and indirect antagonistic mechanisms of the antagonistic yeast strains obtained in this study were evaluated. This study showed that VOC production played a role in the antagonistic activity of
T. indica, W. anomalus and
K. ohmeri against
Cu. lunata,
F. moniliforme,
P. oryzae and
R. solani but not
H. oryzae. Our findings agree with those reports that emission of VOCs by antagonistic yeasts have proven to be one of the important direct antagonistic mechanisms against pathogenic fungi [
27,
28,
29,
30]. In this study, some yeast strains produced the fungal cell wall lytic enzymes β-1, 3-glucanase (12 strains) and chitinase (six strains) in liquid medium, although with relatively low activities. Therefore, the production of β-1,3-glucanase and chitinase seems to be one of the direct antagonistic mechanisms of the antagonistic yeast for inhibition of pathogenic fungi used in this study, which is same as that observed in various antagonistic yeast such as
C. oleophila,
M. guilliermondii and
P. membranifaciens [
22,
31]. Competition for nutrients between antagonists and pathogenic fungi is among the direct antagonistic mechanisms. This mechanism is not easy to demonstrate on plants because it is difficult to control the other mechanisms [
67]. In this study, we tested this mechanism in vitro on PDA with different nutrient concentration. The concentration used in the tests could be different from that available in plants. Unfortunately, we did not check the concentration of nutrients in the rice sheath. The results of the present study showed that the highest fungal mycelial growth inhibition was observed when standard nutrient concentration was used and the efficacy of fugal mycelial growth inhibition decreased when cultured at lower nutrient concentrations. These results could be interpreted to mean that nutrient competition was not a mechanism of these antagonistic yeasts against rice pathogenic fungi. Our study revealed that all of the antagonistic yeasts, except
M. guilliermondii DMKU-RP26, showed the ability to form biofilms when grown in PDB. This ability could be one of the direct antagonistic mechanisms of these antagonistic yeasts. In the present study, we found that only the strains of
W. anomalus produced siderophores. Therefore, this could be one of the antagonistic mechanisms by which
W. anomalus controls the tested rice pathogenic fungi. In this study, strains of
T. indica and
W. anomalus showed phosphate and zinc oxide solubilizing activities. Our result appeared to be the same as that of
T. globosa which exhibited phosphate solubilization to promote plant growth and be one of the biocontrol mechanisms [
21]. Our results indicated that for
T. indica and
W. anomalus, VOC production was the major mechanism, whereas production of β -1,3-glucanase and chitinase, biofilm formation, and phosphate and zinc oxide solubilization were hypothesized as possible additional mechanisms. On the other hand, in the other yeast species, such as
K. ohmeri, M. caribbica and
M. guilliermondii, the antagonistic activity may result from the production of VOCs, β -1,3-glucanase and chitinase and biofilm, however, they did not have the ability to solubilize phosphate and zinc oxide. The antagonistic mechanisms determined in vivo should be further studied.
In this study, the antagonistic yeast cell concentration used in controlling rice sheath blight was 10
8 cells/mL, which was the same as that used by Rosa et al. [
21] to evaluate the efficacy of
T. globosa in controlling anthracnose in sorghum caused by fungus. They reported that this concentration significantly reduced the anthracnose of sorghum. Recently, Khunamwong et al. [
63] reported that it was possible to significantly suppress rice sheath blight disease in a greenhouse by spraying a 10
8 cells/mL of
W. anomalus DMKU-CE52 and
W. anomalus DMKU-RE13. In this study, examination of yeast population on rice sheath surface during controlling rice sheath blight revealed a decrease of population from 7.29–8.74 × 10
4 CFU/cm
2 to 2.51–2.58 × 10 CFU/cm
2 after 5 days of spraying yeast cell suspension. However, the disease lesion development when inoculating
R. solani with yeast was lower when compared to inoculating
R. solani alone. This indicated that a yeast population of 10
2–10
5 CFU/cm
2 was enough to reduce sheath blight disease. However, Fokkema et al. [
68] suggested that at least 10
4 CFU/cm
2 of yeast was necessary to control necrotrophic fungal pathogens on rye and wheat leaves and the higher values of antagonist populations might be required to obtain a better control of decay. The decreasing of yeast population on leaf surface was already found on bean leaves when applied with
R. glutinis or
Cry. albidus [
69] and on wheat leaves applied with
Sp. roseus or
Cry. laurentii [
70].
Sheath blight is the second most important rice disease worldwide after blast [
17,
18]. In this study, strains of
T. indica (two strains) and
W. anomalus (one strain) were evaluated for their ability to control rice sheath blight disease in rice plants in the greenhouse. Although the suppression of rice sheath blight by these antagonistic yeast strains was high, it did not reach the level of efficacy of the chemical fungicide, validamycin. However, using antagonistic yeasts as biocontrol agents in agricultural crops is an environmentally friendly alternative method. Some bacteria, actinomycetes and yeast strains have been found to be capable of controlling rice sheath blight disease in the greenhouse. A strain of
Streptomyces philanthi was found to be effective in the control of rice sheath blight disease in the greenhouse when either spores or a cell suspension was applied [
14].
Sporobolomyces sp. LR951565 was reported to control rice sheath blight disease in the greenhouse [
71]; however, the efficacy of this yeast strain seems to be less than the strains in this study.
W. anomalus DMKU-CE53 and
W. anomalus DMKU-RE13 were reported to control rice sheath blight disease in the greenhouse and the biocontrol efficiency were 55.2–65.1%. This is similar to the efficiency of
W. anomalus DMKU-RP25, the strain in this study (66.4%.) [
65]. To our knowledge, this is the first report of using
T. indica for the biocontrol of rice sheath blight disease caused by
R. solani.