Induced single spore germination in Ganoderma ...

Mushroom Research 19 (2) : 68-73, 2010

Induced single spore germination in Ganoderma lucidum Neeraj Srivastava, Jyoti Pandey, A Verma and Ram Prasad Amity Institute of Microbial Technology, Amity University Uttar Pradesh, Noida (UP)

ABSTRACT Higher basidiomycetes have been used in folk medicine throughout the world since ancient times, as they are known to have antitumour properties. Ganoderma genus is a member of the order polyporales and includes the cultivated mushroom G. lucidum. G. lucidum spores has two hard sporoderms, because of the ingredients silica and calcium mixed into chitin and hard to germinate. For the germination purpose various media compositions were tested and SDWA (Sawdust Water Agar) with 1 & 2% ethyl alcohol resulted 17% spore germination against other medium, which yielded no/low germination. Key Word: Mushroom, polyporales, Ganoderma, spore germination

Mushrooms have been used by man for thousands of years, not only for food, but also for medicinal purposes, as tools, tinder, dyes, perfumes, soap, decoration, magic (Thoen, 1982). Higher basidiomycetes (as of Polyporaceae family) mushrooms have been used in folk medicine throughout the world since ancient times (Wasser and Weis 1999), as they are known to have antitumour, antidiabetic, hyportensive, antiobesity and anti-arteriosclerosis properties. Ganoderma lucidum, called Reishi in Japan and Ling Zhi in China is the most popular medicinal mushroom in China and has been used for a wide range of health benefits like maintenance of health and treatment of chronic and acute ailments. It is known as the 'mushroom of immortality' in Japan and the 'longevity mushroom' in Korea. World trade in Ganoderma lucidum products is in the range of 1.5 billion US $.

Current trade in India is about Rs.120 crore per annum. Though the fruiting body of G. lucidum has been utilized as medicine for several thousand years in China, its spores were recognized and utilized only in the 20th century. Previous studies suggest the spores contain a variety of bioactive components, including polysaccharides, triterpenoids, sterols, proteins, nucleosides, fatty acids and cerebrosides, as in the fruiting body (Yeung et al., 2004). The bioactivity of the spores may be much higher than that of the fruiting body (Min et al.,1998; Zhu et al., 2000; Yu et al., 2009). Ganoderma lucidum is a typical example of the tetrapolar incompatibility system in the fungal class basidiomycetes. All the spores produced during its sexual cycle are self-sterile and shows bifactorial incompatibility. Also the spores produced by the fungus are genetically diverse

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and most of them are always not compatible. The diversity in single spore isolates is also one of the pre-requisite for any breeding program and since the single spores are self-sterile, gene expression in SSIs are not possible. This is only in case of a heterozygous condition where gene expression /characters can be assessed. Hence, breeding followed by a selection is one of the most important factors in G. lucidum. But before the breeding program, one needs the spore germination and the spore germination in G. lucidum is always been a problem. The extremely hard and resilient sporoderm is a barrier for the release of components inside the spores. The aim of the present study is to standardize a method for spore germination in G. lucidum on various media, media supplements, germination inducers, etc. MATERIALS AND METHODS Collection of Spores Spores were collected as per the of method of Carmel et al., (2002) and Ho and Nawawi (1985). Spores were collected by exposing sterile petridishes under sporulating sporophores suspending a plastic plate covered with clean white butter paper 5–10 mm below the surface of the pores of each basidome/fruit body. The entire fruit body and suspended plate were then enclosed in a large sheet of clean paper to minimize air-borne contamination at night. After 24 hours spores prints and basidiomes were collected. Basidiospore Morphology Basidiospore morphology was examined with bright-field and phase contrast microscope. Spores were observed by light microscope techniques using Melzer's Reagent or 2% KOH and phloxine. Spores length, width, and index of each of two wild collected strains of G. lucidum were examined by the method given by Steyaert (1980).

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Preparation of Spore Suspension Serial dilution method was adopted for the preparation of spore suspension. An inoculating loop was flame sterilized and slightly dipped in to autoclaved water and then touched the collected spore prints. Again this loop containing spores was dipped in a drop of sterile distilled water and streaked onto plates containing distilled water agar. Effect of Media/Media Supplement on Germination One ml of the spore suspension was then plated on to petridishes with various media. The plates were inoculated by pour plate method and spread plate method. In spread plate method 1 ml of spore suspension was spread over the medium with the help of sterilized glass spreader. In another pour plate method, 1 ml of spore suspension was put in gamma sterilized plastic petriplates and then media cooled up to 50-55°C poured onto the plates. Six replicates were made for each medium. The medium used in the study were Water Agar, Malt Extract Agar, Potato Dextrose Agar, Combination of MS and Kaefer Media, Water Agar with Biotin, Water Agar with Biotin and meso-Inositol, Water Agar with Biotin and Acetic Acid, Saw Dust Agar with Biotin, Saw Dust Agar with Biotin, acetic Acid, Water Agar with Yeast Extract, Water Agar with Biotin, Rose Bengal, and mycelial block, Water Agar with 1%, 2%, 3% absolute ethyl alcohol. The plates were incubated at 27 ± 1°C and examined after 12, 24, 36, 48, 60 and 72 hrs. Generally the plate contained 25-30 spores and their percentage of germination was recorded. RESULTS AND DISCUSSION Basidiospore Morphology Basidiospores of G. lucidum were brown and ovate, with a truncate to rounded apex, and an eccentric hilar appendix on a rounded base. Spores had a double wall with inter-wall pillars


Fig.1. Measurement of basidiospore morphology

Fig. 2. Few germinating spores of G. lucidum

separating the two walls. The apex contained a germ canal from which germ tubes were observed to emerge. Most spores contained a large vacuole and all were negative in Melzer's reagent. In an average the basidiospores size of G. lucidum varied from 0.2 to 0.3m (Fig.1). The mean of the spore index (length/width ratio) of G. lucidum was 1.50m. Spore Germination Basidiospores of G. lucidum germinated readily if plated less than one week after collection, older spores did not germinate easily and germination rate was too low. Spores germination was not tested further on other rich medium because

contamination was high and water agar was the preferred medium for germination. Water agar is essentially free from nutrients and do not support growth of contaminants, which may interfere with germination by stimulating or inhibitory effects. Various media were tested and the best germination results were obtained with saw dust water agar with 1% and 2% ethyl alcohol (Table 1) (Fig. 2) and found to be the most suitable medium for germination @17%. Good percentage of spores germination was also recorded on SDWA, SDWA + Inositol, SDWA+ Biotin and SDWA + Biotin + Inositol, i.e. 8%, 6.5%, 11%, 7%, respectively but the growth rate of germination tube was much

Table 1. Effect of media on germination of spores Germination of spore (in %) 24 hrs 48 hrs

Media Malt Extract Agar Potato Dextrose Agar Combination of MS and Kaefer Media Czapek Agar Water agar Sawdust water agar SDWA with 1 % ethyl alcohol SDWA with 2 % alcohol SDWA with 3 % ethyl alcohol SDWA with 1 % ethyl alcohol

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0


72 hrs

96 hrs

0 0 0 0 1 1 3 7 9 3

0 0 0 0 1 8 12 18 16 11

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rate of spore germination was too low. These observations lead to the design of another experiment in which the correct dilution of the spores was tested for the optimum spore germination percentage. The results are tabulated in the Table 2.

Fig.3. Plates showing germinated G. lucidum spores

slower on these media when compared to that with SDWA + 2% absolute alcohol. Germination was completely absent or very low in MS, PDA, MEA, Czapek dox agar etc. Mycelial mat on the lid of petridishes was also used as it was reported by many workers that mycelia block produces some volatile substances, which stimulate spore germination. In this study it was also found that petriplate containing mycelial mat on lid invariably stimulates germination process of spores (Fig.3).Also the spore germination was found to be affected by the spore concentration in the plate. It was observed during the study that below a certain concentration of the spores, the

Though the fruiting body of G. lucidum has been utilised as medicine for several thousand years in China, but its spores were recognized and utilised only in the 20th century. G. lucidum spores are tiny and mistlike particles with outer bilayers of sporoderm. Previous studies suggests that the spores contain a variety of bioactive components, including polysaccharides, triterpenoids, sterols, proteins, nucleosides, fatty acids and cerebrosides, as in the fruiting body (Yeung et al., 2004). But the extremely hard and resilient sporoderm is a barrier for the release of components inside the spores. And until the sporoderm is not broken, there are fewer pharmacological effects observed and, on the contrary, the breaking of the sporoderm can improve the release of active components and exertion of their effects (Min et al.,1998). In previous studies, soaking, physical smashing, ultrasonic, high pressure and enzyme hydrolysis methods have been used to break the sporoderm (Li et al., 2004; Ni et al., 2002; Wu et al., 2004; Xie et al., 2006).

Table 2. Spore germination percentage on different dilution Days

Dilutions

Dilutions

10

-2

10

Alcohol conc.

1%

2%

2nd

-

-

4th

-

6th

10

-6

2%

1%

2%

-

-

-

-

-

-

-

-

-

+

-

-

-

-

-

8th

+

+

+

-

-

+

10th

+

+

+

+

+

+

% Germination

8%

12 %

16 %

7%

9%

71

1%

-4

12 %


It is important that the fungal growth, spore release, and spore germination of Reishi occur over a wide range of conditions. This versatility of fungus may explain the ubiquitous occurrence through a wide range of climates from various states in India. The fungus grows well over a wide range of temperature, which may allow it to germinate dormant spore. The ability of the fungus to utilize a wide range of carbon and nitrogen sources may allow it to colonize various tissues of living and dead trees. The environment condition found at the site of collection are similar to those required for germination. The relative humidity in the laboratory climate during cultivation is likely to exceed the 65-75 % required for germination too. Spore germination implies a change from the dormant to an actively growing state. Factors favourable to spore germination may not necessarily be favourable to mycelial growth. Growth of mycelium can sometimes continue under conditions that are inhibitory to germination or vice versa. The optimum temperature for spore germination of Ganoderma lucidum was found to be 27-30°C and the best medium to be saw-dust water agar along with 2% absolute alcohol. Factors affecting spore germination are not well known and some of these have been investigated in this study. The higher rate of germination in saw dust water agar with ethyl alcohol during the present study may be due to the removal of hard sporoderm by ethyl alcohol which ultimately allowed the spores to grow out. It was also observed that the spores swelled slightly prior to germination. Each spore then produced a single germ tube from the truncated end. The results during the present study indicated that germination and mycelial growth was more successful on natural rather than synthetic media such as Czapek dox agar. REFERENCES 1.

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