Breeding and Screening of Lentinula edodes Strains Resistant to Trichoderma spp.

Trichoderma spp. cause large crop losses of the cultivated shiitake mushroom, Lentinula edodes. We bred several shiitake strains that are resistant to Trichoderma spp. using di-mon mating to establish a useful method for controlling the greenmold disease. We examined the competitive ability of L. edodes against Trichoderma spp. using a dual culture system to select resistant strains. By screening Trichoderma-resistant strains, we found that among 11 parental strains, 4 strains, including KFRI 36, were confirmed resistant strains. They showed especially strong resistance to T. harzianum, which formed deadlock after mycelial contact and then invaded into the territory of T. harzianum. KFRI 171 also showed resistance to T. atroviride strains. Among 13 strains, which were made by hybridization of shiitake strains, 5 were confirmed to be resistant to Trichoderma, including KFRI 58-1. Their resistance was not correlated to the resistant activity of their parents' strains. Two strains lose resistance and two strains acquire resistance compared to their parents' strains. In SEM observation, the mycelium of L. edodes at the interaction zone of Lentinula-Trichoderma was rugged and swollen by T. harzianum.

The shiitake mushroom, Lenitula edodes, is widely cultivated and managed throughout Korea. However, a wide range of pests and disease occur in cultivation. Mushroom yields and quality may be reduced by diseases caused by Diatrype sp., Hypocrea sp., Nitschkia sp. (Bak and Kwon, 2005). Among them, fungal species in the genus Trichoderma are commonly existed in soil, which used biocontrol agents of some phytopathogenic fungi. They produce a large range of secondary metabolites which have inhibitory activities, direct mycoparasitism as well as lysis and degradation of cell wall by enzymes (Howell, 2003). However, Trichoderma species, such as T. harzianum and T. polysporum, often attack and kill shiitake mycelium in bed-logs for mushroom by producing antifungal substances and mycolytic enzymes (Seaby, 1998; Tokimoto, 1985; Ulhoa and Perberdy, 1992), thereby reducing the yield of shiitake cultivation. However, control of these diseases was not effective because of the lack of effective control methods. Shiitake cultures produce at least five straight-chain alcohols, which could act as antifungal substances (Ishikawa et al., 2001) and extracellular enzymes, which could help to adapt environmental stress or antagonists (Mata and Savoie, 1998). These substances may play a role in the resistance of Trichoderma spp. through their antifungal effects (Savoie and Mata, 2003; Tokimoto et al., 1987). These days, breeding of shiitake strain is a useful way to acquire strains that are resistant to Trichoderma spp. It can increase their resistance activity by insertion of resistance genes, which produce some antifungal substances (Tokimoto and Komatsu, 1995). In this study, we bred strains of shiitake that are resistant to Trichoderma spp. by di-mon breeding; dikaryon mycelium contact with another monokaryon mycelium that monokaryon is adapted to a donor for cytoplasmic substances to dikaryon mycelium. We then tested them against five strains of Trichoderma spp. to confirm their resistant activities.

Materials and Methods

Test organisms

Lentinula edodes strains were collected from various sources and maintained at the Korea Forest Research Institute (KFRI), Seoul, Korea. The selected shiitake strains (dikaryons, KFRI 36, 38, 57, 58, 59, 171, 182, 183, 192, 193, 194) were bred with monokaryons (KFRI 535, 536), which were isolated from the spores of the KFRI 405 fruiting body. The newly bred shiitake strains were obtained by di-mon breeding method (Table 1). Trichoderma spp., were isolated from KFRI shiitake cultivation and five strains were used in this experiments. All of the fungal cultures were incubated on potato dextrose agar (PDA, Gellix™, Korea) at 23℃ for 10 days and used as inoculum. And all of the experiments carried out five replicates.

Table 1

Hybrid strains made by di-mon mating method

aL. edodes strains maintained in Korea Forest Research Institute

bMonokaryons derived from spores of KFRI 405

cNewly made L. edodes strains by di-mon mating

Dual culture of Lentinula edodes and Trichoderma spp. on agar medium

Competitive interactions between shiitake and mycoparasitic fungi were studied in dual-culture experiments on a PDA in vitro system. In each experiment, 5 mm diameter mycelial disks, which transferred to the shiitake and Trichoderma spp. cultures, placed on the PDA apart from 30 mm. The shiitake inoculum were prepared 7 days before Trichoderma inoculation because of its stabilization before Trichoderma's attack. The shiitake-Trichoderma paired in all possible combinations and five replicates were carried out all the experiments. The fungal cultures were incubated at 23℃ and measured their invading zone, after 7 and 30 days later. The invading zone were divided into 5 types as follow: No resistant activity, L. edodes was completely invaded by Trichoderma spp.; Deadlock, both L. edodes and Trichoderma spp. stop growing after mycelial contact and formed strong antithetic zone line; Weakly resistant, L. edodes partially overgrew the territory of Trichoderma spp.; Moderately resistant, L. edodes overgrew up to the Trichoderma spp. inoculation site; Strong resistant, L. edodes completely overgrew the territory of Trichoderma spp.

Antagonism interactions of Lentinula edodes against Trichoderma spp.

To observe antagonistic action of L. edodes on Trichoderma spp., the two fungi were co-cultured on PDA. After 1 month of incubation, a strong antithetic line was formed on the agar. The L. edodes resistant to Trichoderma spp. were observed by light and scanning electron microscopy. The interacting zones between L. edodes and Trichoderma spp. were cut and prepared for scanning electron microscopic observation (Jacob et al., 1996).

Results and Discussion

Screening of resistance strains against Trichoderma spp.

Among 11 L. edodes strains, only 4 (KFRI 36, 38, 58, 171) were resistant to Trichoderma harzianum (Table 2). At the initial time of incubation, most of L. edodes strains were slightly invaded or formed deadlock with Trichoderma spp. After one month, L. edodes was partially or completely invaded by Trichoderma spp. in most pairings (Fig. 1). However, KFRI 36, 38, 58, 171 showed resistance to T. harzianum and overgrew the territory of mycoparasitic fungi. KFRI 171 also showed resistance to one of T. atroviride strains. KFRI 58 had a similar competitive activity against Trichoderma spp. They stopped growing with the formation of strong antithetic zone lines with T. harzianum and T. atroviride after one month. These resistant strains need to be studied further in bedlog culture and then might be used for greenmold disease resistant strains. L. edodes had some antifungal activities, including antioxidant and enzyme activities. These antifungal activities may be increased their resistant activities against some mushroom pathogenic fungi, for example Trichoderma spp. (Savoie et al., 1998; Savoie and Mata, 2003). The understanding of these particular interaction may be a useful key to the control of greenmold disease during shiitake cultivation and needs further investigation.

Table 2

Resistance of L. edodes strains against Trichoderma species

q±: Deadlock, both L. edodes and Trichoderma spp. stop growing at antithetic zone line, -: No resistant activity, L. edodes was invaded by Trichoderma spp., +: Weakly resistant, L. edodes partially overgrew the territory of Trichoderma spp., ++: Moderately resistant, L. edodes overgrew up to the Trichoderma spp. inoculation site, +++: Strong resistant, L. edodes completely overgrew the territory of Trichoderma spp., A: observed 7 days after incubation with Trichoderma spp., B: observed 30 days after incubation with Trichoderma spp.

Fig. 1

Resistance of L. edodes to T. harzianum (biotype T-8) on PDA (right: T. harzianum, left: L. edodes). T. harzianum invaded into territory of L. edodes (A). Both L. edodes and T. harzianum stop growing, and formed antithetic line (B). L. edodes resistant to T. harzianum, and overgrew into territory of T. harzianum (C).

Screening of resistance strains against Trichoderma spp. made by di-mon mating

Thirteen hybrid shiitake strains were bred by di-mon breeding method (Table 1). Most of the hybrid strains formed deadlock or invading zone at the initial time of incubation, like their parent strains. Among them, only 5 strains showed resistant to Trichoderma spp. KFRI 57-1 and 59-1 had resistance activity against T. reesei and T. harzianum, respectively. However, KFRI 38-1, 38-2 and 36-1 lost the resistance activity of their parent strains against T. harzianum. KFRI 58-1 had resistance activity against T. atroviride but lost their resistance activity against T. harzianum, compared to their parent strains. These results may be associated with insertion of foreign genes of monokaryons derived from basidiospores. On breeding of shiitake strains resistant to Trichoderma spp., Tokimoto and Komatsu (1995) observed that the resistance was revealed dominantly. However, in the new hybrid strains of L. edodes in our study, resistance to Trichoderma spp. was not dominant inherited. The mechanism of these resistant gene transfers might be the key in making resistant strains and needs further investigation.

Interaction between shiitake and mycoparasitic fungi in vitro system

In the interaction zone between L. edodes and Trichoderma spp., the hyphal cell of L. edodes was distorted with local swellings and was inhibited by Trichoderma spp. In untreated L. edodes, the hyphal cell was smooth, straight and normally-growing (Fig. 2). On the Pleurotus-Trichoderma interaction, Reper and Penninckx (1987) observed that mycoparasitic fungus affected the growth of P. ostreatus mainly by producing a volatile toxin which killed P. ostreatus. In our study, L. edodes mycelium were resistant to Trichoderma spp. mechanically by forming brown barrages in the antithetic zone line. Through SEM observation, we determined that the hyphal cells of antithetic zone line were swollen and thickened. This could be associated with the formation of melanin compounds that protect hyphal cells, making them resistant to mycoparasitic fungi attack. Some studies suggest that melanin formation is part of a defensive response against mycelial invasion and that these compounds help fungi to adapt to environmental stress (Rayne et al., 1994; Badalyan et al., 2004). These could be associated with release of volatile and non-volatile metabolites produced by Trichoderma spp. (Howell, 2003).

Fig. 2

Hyphal interactions of L. edodes and T. harzianum (biotype T-8). Hyphal cells of L. edodes. (A) Rugged, (B) swollen and (C) influenced by T. harzianum.

Table 3

Resistance of hybrid L. edodes strains against Trichoderma species

a±: Deadlock, both L. edodes and Trichoderma spp. stop growing at antithetic zone line, -: No resistant activity, L. edodes was invaded by Trichoderma spp., +: Weakly resistant, L. edodes partially overgrew the territory of Trichoderma spp., ++: Moderately resistant, L. edodes overgrew up to the Trichoderma spp., inoculation site, +++: Strong resistant, L. edodes completely overgrew the territory of Trichoderma spp., A: observed 7 days after incubation with Trichoderma spp., B: observed 30 days after incubation with Trichoderma spp.