Gerardo Mata

Extracellular laccase production during hyphal interactions between Trichoderma sp. and Shiitake, Lentinula edodes

Lentinula edodes (Berk.) Pegler was cultivated in liquid media containing malt and yeast extract. Extracellular laccase activity, measured in the culture fluids, was 5–18 times higher in cultures incubated for 29 days than in cultures incubated for 24 days. The addition of water-soluble lignin derivatives or Trichoderma sp. in cultures of L. edodes incubated for 11 days increased laccase activity 3- to 20 fold. The higher response was obtained with live mycelium of Trichoderma sp., but cell-free culture fluids of Trichoderma sp. in pure cultures were also effective. Trichoderma sp. induced changes in the laccase isoenzyme pattern as a result of the alteration of laccases secreted by L. edodes and not the induction of new isoforms.

Waste-reducing cultivation of Pleurotus ostreatus and Pleurotus pulmonarius on coffee pulp: changes in the production of some lignocellulolytic enzymes

Fruiting body production for one strain of Pleurotus ostreatus and three strains of P. pulmonarius was evaluated on coffee pulp pasteurized at 80 °C for 1 h. Based upon three harvests per strain, the single P. ostreatus line was found to display a 40-day culture cycle, whereas the three P. pulmonarius strains completed their cycles after more than 50 days of incubation. These time periods were notably shorter than those observed in previous studies using other growth substrates. Nevertheless, yields expressed as biological efficiencies were not significantly different among strains, fluctuating between 125 and 138%. Extracellular enzymatic activity was also monitored for P. ostreatus and P. pulmonarius (one strain only). To do this, samples of mycelium-bearing substrate were taken every 4 days throughout the incubation period. Care was taken to represent all developmental stages, including primordial and fruiting bodies. Samples were either lyophilized and then analysed or, in some cases, analysed immediately without lyophilization. Hydrolase activity (i.e. endoglucanase (CMC) and cellobiohydrolase (CBH)) was found to depend on developmental stage, showing peak production during fruiting body formation. On the other hand, oxidase activity-(i.e. laccase (LAC) and Mn-peroxidase (MnP)) was associated with phenol degradation. Nevertheless, in the case of oxidases developmental timing differences were also observed. Specifically, LAC activity was detected as early as 8 days after inoculation in non-lyophilized samples, whereas MnP appeared near the end of the incubation period. No LAC activity was observed in lyophilized samples. This study concludes that coffee pulp might be successfully employed in the cultivation of mushrooms, not only because important extracellular enzymes are produced by mushrooms when grown upon this substrate, but also because the abbreviated cultivation cycle associated with this medium favours commercial processes. Commercialization might be further improved if strains specifically adapted to this novel substrate are selected.

Trichoderma harzianum metabolites pre-adapt mushrooms to Trichoderma aggressivum antagonism

Trichoderma spp. is the cause of green mold, a disorder that affects cultivated mushrooms. The aims of the study were to establish whether improvement of mushroom resistance to Trichoderma aggressivum could be obtained by inducing reaction mechanisms before contact with the pathogen and whether this ability was species or strain dependent. Twenty nine isolates of Agaricus bisporus, 29 isolates of Lentinula edodes and 18 isolates of Pleurotus spp. were studied. The effect of T. harzianum metabolites on mycelial growth of these isolates was evaluated on YMEA (yeast, malt extract and agar), supplemented or not with Lysing Enzymes from T. harzianum (Sigma®, L1412). Mycelial growth generally was affected by Lysing Enzymes, but some L. edodes and Pleurotus spp. adapted to Lysing Enzymes. When mycelium was taken from a first culture with Lysing Enzymes and placed on YMEA with Lysing Enzymes for a second culture, their growth rate was not different from those of the controls. In the case of A. bisporus, only partial adaptation was obtained with a few isolates. The effect of adaptation to Lysing Enzymes on resistance to T. aggressivum was assayed for one strain of each group. Trichoderma aggressivum was exposed to the margin of 5- to 9-day-old mushroom colonies. Agaricus bisporus produced brown droplets, and T. aggressivum overgrew its mycelium. Lentinula edodes and P. ostreatus produced brown lines blocking the progression of T. harzianum, both on YMEA and YMEA plus Lysing Enzymes. The line was visible after 3 d on YMEA and after only 2 d on YMEA plus Lysing Enzymes. Improvement in the resistance to antagonists by introduction of some of their metabolites to the culture medium is a method for mushroom protection.

Role of Bacillus spp. in antagonism between Pleurotus ostreatus and Trichoderma harzianum in heat-treated wheat-straw substrates.

This study aimed to identify bacteria involved in Trichodermaharzianum inhibition while promoting Pleurotus ostreatus defences in order to favour cultivation-substrate selectivity for mushroom production. PCR-DGGE profiles of total DNA from wheat-straw substrate showed weak differences between bacterial communities from substrate inoculated with P. ostreatus with or without T. harzianum. The major cultivable bacteria were isolated from three batches of wheat-straw-based cultivation substrates showing an efficient selectivity. They were screened for their ability to inhibit T.harzianum. By using specific media for bacterial isolation and by sequencing certain 16S-rDNA, we observed that Bacillus spp. were the main inhibitors. Among them, a dominant species was identified as Paenibacillus polymyxa. This species was co-cultivated on agar media with P. ostreatus. The measurement of laccase activities from culture plugs indicated that P. polymyxa induced increases in enzyme activities. Bacillus spp. and specifically P. polymyxa from cultivation substrates are implicated in their selectivity by both inhibiting the growth of T.harzianum and stimulating defences of the mushroom P. ostreatus through the induction of laccases. The management of microbial communities during P.ostreatus cultivation-substrate preparation in order to favour P. polymyxa and other Bacillus spp. growth, can be a way to optimize the development of P. ostreatus for mushroom production or other environmental uses of this fungus.

Interspecific interactions with Trichoderma longibrachiatum induce Pleurotus ostreatus defence reactions based on the production of laccase isozymes

Laccases are phenoloxidases involved in aromatic compound transformation but also in stress response towards antagonist species such as Trichoderma sp. In this study intracellular isoforms of laccases produced by Pleurotus ostreatus in liquid cultures with or without Trichoderma longibrachiatum showed five isoforms with various intensities depending on the culture conditions suggesting a basal expression of these enzymes, which can be induced by interspecific interactions. A first attempt to analyse the induction of P. ostreatus laccase-gene expression by a biotic factor was realized using semi-quantitative RT-PCR. We showed that the transcription of a laccase gene of P. ostreatus can be modified by a biotic stress such as T. longibrachiatum.

Viability and mushroom production of Lentinula edodes and L. boryana strains (Fungi: Basidiomycetes) after cryogenic storage of spawn stocks

The viability of two strains of Lentinula edodes and two of L. boryana under cryogenic storage during 1 week has been studied from the evaluation of five contact periods (1, 1.5, 2, 3 and 5 h) of the cryoprotector, glycerol 10% (v/v), with the mycelium. On average, 99.25% of samples were recovered, 1.5 h being the best contact period. Afterwards, samples of the strains, before and after the cryogenic process, were cultivated at a pilot plant using a mix of Carpinus carolineana sawdust, rice bran and sorghum grains as substrate. The evaluation parameters were: days of incubation, primordia initiation, number of flushes, fruiting body sizes and biological efficiency (EB). Only L. edodes developed carpophores. On average, 3–4 flushes were obtained, which reached EB of 67.1 ± 30.7 to 74.7 ± 24.5 with no statistical differences detected between the yields. The majority of fructification sizes ranged from 5 to 14.9 cm. Morphological differences between the samples before and after the treatment were not observed.

Preservation of Agaricus subrufescens strains at low temperature by using cultures on sorghum grains.

BACKGROUND One of the main problems for the preservation of genetics resources of Agaricus subrufescens is to maintain the viability of the strains because the mycelium is very sensitive to cooling and therefore it ages rapidly. AIMS Evaluate the viability of A. subrufescens strains stored as cultures on sorghum grain (spawn) at different temperatures. METHODS Eighteen strains of A. subrufescens and three strains of Agaricus bisporus were studied. Spawns viability was evaluated under the following conditions: (1) control at 25°C (C), (2) cooling to 4°C (R) and (3) freezing in liquid nitrogen at -196°C (LN). Samples were recovered from week 4 every 2 weeks until week 12 and week 24 in C and R, whereas in LN samples were recovered at 4, 12 and 24 weeks. Viability was evaluated in 50 seeds, by strain and condition, recovering the mycelium in Petri dishes with potato dextrose agar medium (PDA). Mycelium growth was also evaluated on PDA after 14 days of recovery. RESULTS Most strains showed 100% viability and they were recovered usually in 1 day. In LN the viability ranged between 84 and 100% depending on the strain, but in some cases recovery took more than 10 days. Mycelial growth decreased gradually over time and although the results show significant differences between treatments C and R, the decline is associated with ageing of the mycelium rather than the treatment itself. CONCLUSIONS Culture on sorghum grain and storage at low temperature is an interesting way to preserve genetic resources of A. subrufescens.

Hydrolytic enzyme activities in shiitake mushroom (Lentinula edodes) strains cultivated on coffee pulp

Hydrolytic enzyme production (cellulases, laminarinases and xylanases) was studied in cultures of Lentinula edodes on sterilized coffee pulp. Samples of substrate colonized by mycelia were taken after 7, 14, 21, 28 and 35 days of incubation at 25°C (W1 to W5) and during the fruiting period at different stages: formation of primordia (PF), first harvest (H) and one week after the first harvest (PH). The enzymatic activity was lower during the early mycelial growth and showed higher levels during the formation and development of fruiting bodies. During the reproductive stage of the fungus, the samples were subjected to a soaking treatment; however, it was not possible to relate this soaking treatment to the increase in enzyme production. The levels of enzymatic activity suggest that secretion of the studied enzymes does not influence the adaptability of the strains to the substrate.

Genetic characterization, evaluation of growth and production of biomass of strains from wild edible mushrooms of Lyophyllum of Central Mexico

The present study conducted a genetic characterization and determined growth rate and biomass production in solid and liquid media, using strains obtained from wild edible sporomes of Lyophyllum that grow in high mountains. Vegetative isolation was used to obtain a total of four strains, which were divided into two clades within the section Difformia: Lyophyllum sp. and Lyophyllum aff. shimeji. Growth rate and biomass production were influenced by both the culture media and the strains. In a potato dextrose agar medium, the strains presented a higher growth rate, while in a malt extract-peptone and yeast agar medium, the growth rate was lower, but with a higher biomass production that was equal to that in the malt extract-peptone and yeast liquid medium.

New Prospects in Pathogen Control of Button Mushroom Cultures

Abstract Cultivated mushrooms are susceptible to a wide range of viral, bacterial, and fungal pathogens. Due to new regulations and expectations of consumers, the conventional ways of controlling them using pesticides, when these exist, have to be replaced by alternative methods. The most promising alternatives are improvement in prophylaxis and in pathogen detection for a better definition of control strategies, development of biocontrol agents and biomolecules, and selection of resistant strains of cultivated mushrooms. This chapter presents an outline of recent developments in this field for the button mushroom Agaricus bisporus .