Induction of sporulation in plant pathogenic fungi

Abstract

Spore morphologies are a major character in fungal taxonomy, although many isolates are not able to sporulate on common artificial media. This article reviews the effect of nutrition, host tissue, and light on fungal sporulation in artificial media. A trial experiment using 42 strains that failed to sporulate on potato dextrose agar (PDA) and half-strength PDA after 3 months is reported. Five strategies (1/10-strength PDA, CaCO₃ water agar, pine needle medium, mulberry agar, and near-ultraviolet light irradiation) were applied to induce these strains to sporulate, with an overall success rate of 62%. Pine needle medium was the most successful method, which induced sporulation of 40% of recalcitrant strains.

Keywords:

• conidia
• pine needle medium
• sterile
• taxonomy

Introduction

Fungal spores are specialized cells that function as resting or dispersal propagules, which are important for fungal dissemination and propagation (Kirk et al. 2008). Spores and fruiting structures are the most important morphological characters used by mycologists to classify fungi into genera or differentiate closely related species. Although DNA sequence data have been popularized in fungal taxonomy, morphological characters are still essential in taxonomy (Hyde et al. 2010). However, numerous isolates fail to sporulate on commonly used media. For example, in endophyte studies, the sterile mycelia account for 4.5–54% of the total isolates (Fisher et al. 1994; Guo et al. 1998; Sánchez Márquez et al. 2008; Sun et al. 2012).

Fungal sporulation is a complex process influenced by environmental and endogenous biological rhythms (Wright 1979; Timberlake 1980). The molecular mechanisms of the induction of conidia in fungi have been intensively studied, mainly in model organisms such as Aspergillus nidulans and Neurospora crassa (Roncal and Ugalde 2003; Sun et al. 2012). A number of genes have been found to be involved in this process, affecting different steps of the signal transduction pathway (Roncal and Ugalde 2003; Xu et al. 2012; Zhou et al. 2012). In addition, numerous interdependent events must interact, both spatially and temporally, during the course of sporulation, e.g., substrate levels and in vivo enzyme activities (Timberlake 1980; Hernandez-Rodriguez et al. 2012; Tzima et al. 2012). Finding effective environmental factors that induce sporulation is often the first step towards making an accurate identification.

This article summarizes the major factors that induce sporulation, including nutrients, host tissues, and light (Smith and Berry 1974; Guo et al. 1998). A trial experiment was conducted using five different methodologies and the results are compared and discussed.

Factors related to fungal sporulation

Nutritional control

Many nutritional factors are effective in fungal sporulation, e.g., the carbon source, nitrogen source, and microelements (Timnick et al. 1951). Some fungi have specific carbon and nitrogen requirements for sporulation (Engelkes et al. 1997; Gao et al. 2007). Potato dextrose agar (PDA) is the most widely used medium in fungal isolation and culture. Other commonly used media include potato carrot agar, malt dextrose agar, yeast extract-phosphate medium, Czapek yeast autolysate agar, malt extract agar, cornmeal agar, potato sucrose agar, V8 vegetable juice agar (Booth 1971). These media provide favorable growth condition for most endophytic and pathogenic fungi, but are often ineffective in inducing sporulation of sterile isolates (Guo et al. 1998; Li et al. 2007).

Sporulation usually occurs when growth rate is reduced and is hampered under conditions that favor rapid mycelial growth (Dahlberg and Etten 1982). Starvation or nutritional depletion often stimulates sporulation, and some modifications of artificial media with low nutrient have been applied (Wulandari et al. 2009; Braun et al. 2011). Some typical low nutrient media include water agar media, half- or 1/4-strength PDA (Masangkay et al. 2000), and synthetic nutrient-poor agar medium (Nirenberg 1976).

Slide culture (Figure 1) (Riddell 1950) is often used in the induction of appressoria and conidiophores in Colletotrichum (Cai et al. 2009; Liu et al. 2011; Su et al. 2011). Slide cultures are also a recommended method for conidial induction (Figure 1). First, the slide was supported by two sticks above one or two layers of filter paper on the bottom of a petri dish, which were autoclaved altogether. Second, the filter paper was soaked with sterilized water to maintain humidity. Third, two 0.5 mm × 0.5 mm pieces of PDA were placed on the slide and inoculated with a small quantity of mycelium at one corner of the agar block. Finally, a sterilized coverslip was placed on the agar block. The assembly was sealed and incubated at the optimum temperature for fungal growth for 7–14 days, after which the coverslip can be removed and observed microscopically.

Figure 1. Slide culture technique to induce the formation of spores. (A) Sterilized filter paper, sticks, and slide. (B) Add sterilized water on the filter paper to maintain humidity. (C) A small amount of mycelia was inoculated on 0.5 mm × 0.5 mm PDA agar. (D) Cover a sterilized coverslip on the agar block, seal the petri dish, and incubate at the fungal growth temperature for 7–14 days. The coverslip was taken out and observed microscopically.

In spite of the fact that nutrient limitation is one of the most widely used strategies to induce conidial formation, its signaling mechanism remains to be understood. One possible explanation is the existence of sensors that signal nutrient status to the cell (Özcan et al. 1996; Roncal and Ugalde 2003).

Host tissue

Plant pathogenic fungi are often used to induce sporulation on the source host tissues; for example, Photita et al. (2001) used banana petioles to induce sporulation of endophytes isolated from wild banana (Musa acuminata) leaves. Autoclaved leaves of Rhododendron pulchrum cv. Ohmurasaki were also found to induce sporulation of Guignardia endophyllicola (Okane et al. 2001). A sterilized petiole fragment of the palm Livistona chinensis was used to induce sporulation of endophytic sterile mycelia isolated from L. chinensis (Guo et al. 1998).

Some particular plant tissues are effective in inducing sporulation. Autoclaved pine needles were added onto the surface of agar media to induce pycnidia of some Botryosphaeriaceae spp. (Crous et al. 2006). Sterilised branch segments of Sophora japonica were utilized to promote sporulation of the endophytic fungi from lichens (Li et al. 2007). Carnation (Dianthus caryophyllus) leaves were found to be a good substrate for sporulation in Fusarium and Pestalotiopsis species (Fisher et al. 1982; Wei et al. 2006; Liu et al. 2010). Leaves of Hydrangea or Gardenia were found to be effective for perithecia and ascospore formation of Glomerella cingulata, Mycosphaerella allicina, Metasphaeria sp., Guignardia sp., and Pleospora herbarum (Furukawa and Kishi 2002). Yoshida and Shirata (2000) showed that the leaves of 22 plant species induced sporulation in Colletotrichum dematium. They also tested compounds in mulberry leaves and found that biotin may play a role in inducing sporulation. The biotin in the plant tissues may alter the synthesis of wall polysaccharides and oleic acid and thus trigger the selective expression of genes involved in sporulation (Leaver et al. 1947; Tomiyama et al. 1962; Misawa 1965; Yamaguchi 1974; Timberlake 1980).

In addition, some chemical supplements also increased sporulation; for example, CaCO₃ induced sporulation of some Alternaria species (Shahin and Shepard 1979; Masangkay et al. 2000). Externally bound calcium has been shown to be directly involved in the induction step (Ugalde and Pitt 1986; Ugalde et al. 1990).

Light effect

Light has been regarded as an important stimulus for conidial formation. Exposure of mycelia to near-ultraviolet (UV) light and blue light has been widely used (Marsh et al. 1959; Dahlberg and Etten 1982; Betina 1995; Xu et al. 2009). For example, spores have been successfully induced by near-UV light irradiation in some species of Ascomycetes (Crous et al. 2006), Basidiomycetes (Miller 1967), Myxomycetes (Starostzik and Marwan 1995), and Zygomycetes (Idnurm et al. 2006).

Wavelengths spanning 350–500 nm are most effective in inducing sporulation (Dahlberg and Etten 1982). Various isolates showed different threshold dosages under particular wavelengths (Rakoczy 1965), and excessive irradiation can inhibit sporulation (Rakoczy 1963, 1965). One commonly used method is 12 hours near-UV light irradiation and irradiation in darkness for 12 hours, imitating the diurnal cycles of light and darkness (Marsh et al. 1959; Leach 1962).

The mechanism by which fungal conidiation is induced by light remains unknown. Dahlberg and Etten (1982) proposed that photoreceptors affect the electron transport system, affecting the pH and ionic balance of the cell as well as glucose and carbohydrate metabolism. Several light-sensing proteins have been identified during the process of fungal conidiation (Corrochano 2007; Idnurm et al. 2010; Rodriguez-Romero et al. 2010; Ruger-Herreros et al. 2011). For example, the White Collar-1 and Vivid proteins have been recently identified as the blue light photoreceptor that mediates light induction of the rhythmic conidiation of N. crassa (Froehlich et al. 2002; He et al. 2002; Schafmeier and Diernfellner 2011). In addition, some transcription factors, e.g., BLR-1 and BLR-2, are vital to the photoconidiation process (Sanchez-Arregui et al. 2012).

Other factors

Though previous studies focused on factors that affect sporulation, such as temperature (Prasad et al. 1973), pH (Yazdany and Lashkari 1975), humidity (Paul and Munkvold 2005), and mutilation of the mycelium (Rands 1917), these studies often focused on spore yield. Generally, combinations of these factors were often adopted, e.g., low nutrient media plus near-UV light irradiation (Crous et al. 2006; Wulandari et al. 2009; Glienke et al. 2011) (Table 1).

Table 1. Environmental stimuli for the induction of conidiation in some important pathogenic fungi

Family or genus	Representative species	Environmental stimulus	References
Alternaria	Alternaria spp.	Nutrient limitation	Rotem (1994)
	A. tomato	Temperature	Aragaki (1961)
	A. cichorii, A. solani	Near-UV light irradiation	Charlton (1953), Vakalounakis and Christias (1981)
		Desiccation	Rotem (1994)
	A. alternata, A. dauci, A. solani	S-medium (water agar amended with CaCO3 and sucrose)	Shahin and Shepard (1979), Masangkay et al. (2000)
	A. solani	Scraping	Singh (1967)
Botryosphaeriaceae	Botryosphaeriaceae spp.	2% water agar with sterilized pine needles + near-UV light irradiation	Crous et al. (2006), Wulandari et al. (2009), Glienke et al. (2011)
	Guignardia mangiferae, Phyllosticta capitalensis, P. citricarpa	OMA + near-UV light irradiation	Glienke et al. (2011)
	Phyllosticta spp.	Near-UV light irradiation	van der Aa (1973)
	Phomopsis phaseolorum	Sterilized plant parts exposed to cool-white fluorescent light	Onesirosan (1978)
Fusarium	Fusarium culmorum	Reduced nutritional levels	Larmour and Marchant (1977)

A trial experiment for induction of sporulation

Forty-two plant pathogenic fungal strains that did not sporulate on PDA and half-strength PDA after 3 months were selected for the experiment. Four media were chosen, namely 1/10-strength PDA (potato 20 g, boiled for 30 min and filtrated; agar 15 g; distilled water added to constant volume of 1 L), CaCO₃ water agar (agar 15 g; CaCO₃ 30 g; distilled water 1 L, pH 7.4) (Shahin and Shepard 1979), pine needle medium (prepared by putting 250 g of fresh pine needles and 20 g of potatoes 20 g into 1 L water, boiling for 30 min, filtrating, keeping the volume to 1 L by adding water, and then adding 20 g of agar), and mulberry agar (mulberry leaf 33 g; sucrose 20 g; agar 18 g; distilled water 1 L) (Yoshida and Shirata 2000). The fungi were identified by morphological characters and phylogenetic analysis.

Twenty-one out of the total 42 tested isolates produced spores (Table 2). Of these, six isolates sporulated on mulberry agar, fourteen on pine needle medium, nine on 1/10-strengh PDA medium, and six on CaCO₃ water agar. The remaining cultures that did not sporulate were exposed to near-UV light, and another five produced spores (Table 2). Thus, 26 out of 42 strains (62%) sporulated.

Table 2. Twenty-six successful sporulation-inducing isolates

			Medium
Host	Strain no.	Preliminary identification	Mulberry agar	Pine needle extracts plus 1/10-strengh PDA medium	1/10-strengh PDA medium	CaCO3 water agar
Trachycarpus fortunei	D3-1	Colletotrichum sp.	√		√
Fern sp.	D2-1	Colletotrichum sp.		√	√
Fern sp.	D2-2	Colletotrichum sp.	√ (combined with near-UV irradiation)
Terminalia arjuna	h8-1	Colletotrichum sp.		√	√
Orchidaceae sp.	h86-1	Colletotrichum sp.		√
Orchidaceae sp.	h86-2	Colletotrichum sp.	√			√
Clinacanthus nutans	h53-1	Colletotrichum sp.		√		√
Viburnum odoratissimum	h41-2	Colletotrichum sp.		√
Camellia sinensis	h6-2	Colletotrichum sp.		√	√
Ophiopogon japonicus	h50-2	Colletotrichum sp.		√	√
Orchidaceae	h79-1	Colletotrichum sp.	√		√	√
Michelia figo	h90-1	Colletotrichum sp.		√ (combined with near-UV irradiation)
llex triflora	DY6-2	Colletotrichum sp.		√ (combined with near-UV irradiation)
Gramineae sp.		Colletotrichum sp.		√
Syzygium jambos	h7-1	Celoporthe sp.	√
S. jambos	h7-2	Cryphonectria sp.	√	√
Cymbidium bicolor	h69-1	Phomopsis sp.		√	√	√
Syzygium tephrodes	h18-1	Endomelanconiopsis sp.				√
Cymbidium sp.	h66-1	Fusarium sp.	√	√
Trachycarpus fortunei	D3-1	Microdochium sp.			√
Areca triandra	h30-1	Phomopsis sp.				√
Viburnum odoratissimum	h41-3	Phomopsis sp.		√	√
Caryota ochlandra	h10-2	Phyllosticta sp.		√
Billbergia pyramidalis	h77-2	Mycoleptodiscus sp.		√
B. pyramidalis	h77-1	Mycoleptodiscus sp.	√ (combined with near-UV irradiation)
Trachycarpus fortunei	h34-1	Pestalotiopsis sp.		√ (combined with near-UV irradiation)

In this experiment, low nutrient media amended with host tissue induced sporulation in many strains. Pine needle medium was shown to be very effective. Similarly, Crous et al. (2006) used tap water agar with autoclaved pine needles to induce sporulation of Botryosphaeriaceae spp. The media containing ground Austrian pine needles successfully induced pycnidia in Diplodia scrobiculata and D. pinea. One hypothesis is that unidentified compounds in pine needles may stimulate sporulation.

Our experiment also showed that both mulberry leaf and pine needle media were effective in inducing sporulation of Fusarium sp. and Celoporthe sp., which produced abundant conidia (Table 2).

Fourteen Colletotrichum strains sporulated in this experiment. Sangeetha and Rawal (2008) tested the best carbon and nitrogen sources required for growth and sporulation of C. gloeosporioides from mango and found heavy sporulation was observed if maltose and ammonium nitrate were used as carbon and nitrogen sources, respectively. Suzaki (2011) compared the conidial mass of C. gloeosporioides on PDA, oatmeal agar (OMA), and carrot juice agar and found that C. gloeosporioides sporulated when the isolates were pre-cultured in potato dextrose broth and then spread on diluted OMA under continuous light. The aims of these studies were to determine the optimal growth and maximal conidial yield of the isolates. Our result indicated that low nutrient is helpful in inducing sporulation in Colletotrichum species.

Onesirosan (1978) studied the factors affecting sporulation of Phomopsis phaeolorum and found that various common media in combination with techniques such as exposure to near-UV light and scraping of the culture surface were ineffective in inducing sporulation, while sterilized plant parts in combination with techniques such as exposure to cool-white fluorescent light were effective in inducing sporulation. In our experiment, three Phomopsis isolates were induced successfully, one only produced spores on CaCO₃ water agar, one on pine needle medium and 1/10-strength PDA, and another one on 1/10-strength PDA, pine needle medium, and CaCO₃ water agar. The results showed that low nutrients, CaCO₃, and pine needle induced sporulation in some Phomopsis strains. Overall, pine needle medium was most effective in inducing sporulation in recalcitrant fungi.

Sterile mycelia are often encountered in taxonomic and ecological studies (Suryanarayanan and Vijaykrishna 2001; Su et al. 2010; Tejesvi et al. 2011; Sun and Guo 2012). The induction of sporulation allows confident identification, which results in a better understanding of the fungal communities in nature. Factors such as low nutrient media, host tissue, and light irradiation should be considered in the induction of sporulation in artificial media.

Acknowledgements

This work was financially supported by Chinese Academy of Sciences (KSCX2-YW-Z-1026) and the National Natural Science Foundation of China (NSFC 31110103906).