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#1 WolfWhiz

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Posted 14 April 2019 - 05:08 AM

Efficiency of treatments for controlling Trichoderma spp during spawning in cultivation of lignicolous mushrooms
Introduction

Trichoderma sp, also known as green mold, is a cellulolytic filamentous fungus, which frequently contaminates mushroom substrates. This fungus is often observed in the early stages of the process, especially during spawning run period, but also during cropping period and causes huge losses in mushrooms crops (Jandaik and Guleria, 1999Yarma and Vijay, 1996).

Trichoderma spp infection in edible basidiomycetes have been known for a long time (Komon-Zelazowska et al., 2007). An Agaricus green mold disease started in Northern Ireland in 1985 and rapidly spread over farms across Europe and was quickly succeeded by subsequent outbreaks all along Ireland in 1986, in England and Scotland in 1987, in the Netherlands in 1994, in France in 1997 and in Spain in 1998 (Hermosa et al., 1999Mamoun et al., 2000). This disease also occurred in the United States and Canada (Castle et al., 1998) causing important economic losses. The escalation of green mold evoked extensive research efforts to identify and study the causative agent (Castle et al., 1998Krupke et al., 2003Hatvani et al., 2007).

Green mold causes economic losses not only in Agaricus but also in Pleurotus and Lentinula cultivation. Sharma and Vijay (Sharma and Vijay, 1996) reported a green mold attack in oyster mushroom in North America. Serious cases of green mold diseases in P. ostreatus in mushroom farms were recently detected in South Korea, Italy, Hungary and Romania (Hatvani et al., 2007). Komón-Zelazowska, et al. (2007), determined that the causal agents of this disease were two genetically closely related, but phenotypically strongly different, species of Trichoderma, which have been described as Trichoderma pleurotum and Trichoderma pleuroticola. They belong to the Harzianum clade of Hypocrea/Trichoderma which also includes Trichoderma aggressivum, the causative agent of green mold disease of Agaricus. Both species have been found on cultivated Pleurotus and its substratum in Europe, Iran, and South Korea, but T. pleuroticola has also been isolated from soil and wood in Canada, the United States, Europe, Iran, and New Zealand. T. pleuroticola displays pachybasium-like morphological characteristics typical of its neighbors in the Harzianum clade, whereas T. pleurotum is characterized by a gliocladium-like conidiophore morphology which is uncharacteristic of the Harzianum clade. Different species of Trichoderma spincluding T. viridaeT. harzianum and T. polysporum, commonly cause also injury both to mycelia and basidioma of Lentinula edodes (Tokimoto and Komatsu, 1979). Trichoderma sp produces several enzymes involved in degradation of the fungal cell walls that may contain chitinases and glucanases (Sivan and Chet, 1989Geremia et al., 1993Ait-Lahsen et al., 2001).

Various treatments are used for the preparation of substrate for mushroom cultivation to eliminate competitive fungi. They are: steam sterilization, steam pasteurization, hot water immersion and chemical treatment (Jaramillo and Albertó, 2013); but they are not always successful. Contaminations according to mushroom growers may even occur sporadically after these treatments during handling or spawning. The purpose of this paper is to estimate the growth of the green mold Trichoderma sp on lignocellulose substrates after different disinfection treatments to know which of them are more effective to avoid contamination during spawning phase.

Materials and Methods
Strains

Strains used in this work are conserved in ICFC (IIB-INTECH collection of Fungal Cultures, Laboratory of Mycology and Mushroom cultivation, IIB-INTECH; Chascomús, Argentina (reference in the WDCM data base: 826).

 

Pleurotus ostreatus

ICFC 153/99, commercial strain, 19-XI-1999, leg. E. Albertó; Gymnopilus pampeanus: ICFC 444/01, Capital Federal, Buenos Aires, Argentina; growing on Eucalyptus, 10-V-2001, leg E. Albertó; ICFC 548/03, Burzaco, Buenos Aires, Argentina, growing on Melia azedarach stalk, 15-11-2003, E. Albertó; Trichoderma sp: ICFC 767/12, Chascomús, Buenos Aires, Argentina; 12-VIII-2012. Leg. M. B. Colavolpe, isolated from P. ostreatus cultivation bag.

Spawn production

It was prepared following Pieckenstain et al. (1999). Briefly, glass bottles filled with boiled wheat grains and 1% w/w CaCO3 were sterilized for 1.5 h at 121 °C, cooled and inoculated with an agar plug (1 cm diam.) cut from the advancing margin of a 5-d-old colony grown on PDA (potato dextrose agar). Bottles were incubated in the dark, at 25 °C, with periodical shaking, during 15 days for Pleurotus and Trichoderma sp, and 30 days for Gymnopilus.

Trichoderma sp inoculum

Conidia of Trichoderma sp were massively produced using the spawn method. When spawning run period was finished, colonized bottles were left near a window at room temperature until the grains became all green colored due to the mature conidia. A suspension of Trichoderma sp conidia in distilled water was prepared. Colonized grains were immersed in distilled water under laminar flow; conidia were released by shaking. Then, water was filtered using cheesecloth and dropped into an Erlenmeyer flask. Concentration of conidia was adjusted to 105 conidia/mL using a Neubauer chamber. Conidia suspension was not stored; a new suspension was made evey time it was needed.

Substrate preparation

Chopped wheat straw, wheat seed, Populus or Eucalyptus sawdust were separately used. Substrates were subjected to different treatments (see next point), then immediately bagged in polypropylene bags (15 × 30 cm) containing 100 g of wet substrate with the addition of 1% w/w of CaCO3. After they reached room temperature, they were sprayed with 3 mL of water suspension of conidia of Trichoderma sp. per bag and mixed. Then, bags were inoculated with 10% of spawn of the mushroom used in each experiment (w/w) and afterwards they were stopped with cotton plugs held by PVC (polyvinyl chloride) cylinders.

Treatments of the substrate

Different treatments were carried out, 1) immersion in hot water: a thermal bath with automatic temperature regulation was used; substrates were placed during 30 min into the bath after reaching the temperature of treatment (60 or 80 °C), then they were drained and put on absorbent papers to adjust the humidity to 70% and finally bagged: 2) steam sterilization, substrates were firstly bagged, then tap water was added up to 70% of final humidity and sterilized during 2 h at 120 °C at 1.2 psi of pressure; 3) Immersion in alkalinized water: we used the methodology proposed by Contreras et al. (2004). Substrates were soaked into an alkaline solution prepared with 0, 5% of calcium oxide for 0, 5 min, 12 h, 24 h or 36 h.

Experimental design and cultivation conditions

Three experiments with different objectives were carried out. The first experiment was focused on the evaluation of the growth of the green mold Trichoderma sp after the steam sterilization treatment. Two strains and four different sterilized and non-sterilized substrates were used (Table 1); the following treatments were performed: T (Trichoderma sp spray inoculum), T + M (Trichoderma sp spray inoculum + mushroom spawn), and M (mushroom spawn). Table 1 shows the experimental design. The second experiment was focused on the evaluation of the effect of different temperatures of heat treatments of substrates on the growth of the green mold. Temperatures of 60, 80 °C (direct immersion in hot water) and 120 °C (steam sterilization of bagged substrates) were assayed. Two strains and three different substrates were used. The following treatments were performed: T, T + M and M (Table 2). The third experiment was focused on the evaluation of the growth of the green mold after immersion of the substrate in alkalinized water. Wheat straw was immersed in an alkaline solution during four different periods of time: 5 min, 12 h, 24 h and 36 h. One strain of Pleurotus ostreatus and one substrate (wheat straw) were used. Three treatments were tested: T, T + M and M. Controls without inoculating were performed in all experiments, bags were observed after 10 days of incubation at 25 °C in the dark. Five replicates for all treatments and experiments were carried out. The results were qualitatively expressed by assessing the visual degree of growth and colonization of Trichoderma sp. The following symbols indicate degrees of growth of the green mold disease in the bags: (+): Poor growth, less than 20% of substrate colonization; (+ +): Intermediate growth, 20–50% of substrate colonization; (+ + +): abundant growth, more than 50% of substrate colonization; (−): non-growth. Figure 1 shows the visual differences in Trichoderma sp growth in the bags with wheat straw substrate spawned with Pleurotus ostreatus and the symbols used.

 
bjm-45-1263-g001.jpg

Growth of Trichoderma sp in the bags: A: (−): non-growth. B: (+): poor growth, up to 20% of substrate colonization; C: (+ +): intermediate growth, 20–50% of substrate colonization; D: (+ + +): abundant growth, more than 50% of substrate colonization.

Table 1

Experimental design to evaluate the growth of green mold disease after sterilization treatment.

  Treatments*   Substrates S NS     T T + M M T T + M M Wheat straw 767/12 767/12 + 153/99 153/99 767/12 767/12 + 153/99 153/99 Wheat seed 767/12 767/12 + 153/99 153/99 767/12 767/12 + 153/99 153/99 Populus sawdust 767/12 767/12 + 548/03 548/03 767/12 767/12 + 548/03 548/03 Eucalyptus sawdust 767/12 767/12 + 548/03 548/03 767/12 767/12 + 548/03 548/03

ICFC strains used: 767/12, Trichoderma sp; 153/99, Pleurotus ostreatus; 548/03, Gymnopilus pampeanus. T: spray of Trichoderma conidia suspension; M: mushroom spawn, S: sterilized substrate, NS: non-sterilized substrate. Five replicates for each test were performed.

*Controls of S and NS treatments without inoculation were also carried out.
Table 2

Experimental design to evaluate the growth of green mold disease after immersion of substrates in water at 60, 80 °C or steam sterilization.

  Treatments*   Substrates 60 °C 80 °C NS S     T T + M M T T + M M T T + M M T T + M M Wheat straw 767/12 767/12 + 153/99 153/99 767/12 767/12 + 153/99 153/99 767/12 767/12 + 153/99 153/99 767/12 767/12 + 153/99 153/99 Populussawdust 153/99 767/12 + 153/99 153/99 767/12 767/12 + 153/99 153/99 767/12 767/12 + 153/99 153/99 767/12 767/12 + 153/99 153/99 Populussawdust 444/01 767/12 + 444/01 444/01 767/12 767/12 + 444/01 444/01 767/12 767/12 + 444/01 444/01 767/12 767/12 + 444/01 444/01

ICFC strains used: 767/12, Trichoderma sp; 153/99, Pleurotus ostreatus; 444/01, Gymnopilus pampeanus. T: spray of Trichoderma conidia suspension; M: mushroom spawn, S: sterilized substrate, NS: non-sterilized substrate. Five replicates for each test were performed.

*Controls of 60 °C, 80 °C, NS and S treatments without inoculation were also carried out.
Results and Discussion

It is well known that substrate is one of the most important contamination sources for green mold disease, especially if it has a high level of carbohydrates (Fletcher et al., 1986). Different species of Trichodermacan contaminate the substrates; this may be due to the use of different substrates, the origin, and manufacturers (Komon-Zelazowska et al., 2007). Contamination is the result of the inoculum potential plus the ability to rapidly grow in the substrate. The treatments of the substrates are generally used to affect the inoculum potential with the objective of eliminating all the spores of Trichoderma spp present in the substrate, but they do not deal with the colonization ability if a new inoculum is introduced after heat treatment. Arrival of inoculum during spawning is frequent, and in a substrate without competitors, this inoculum may develop rapidly. It is very common for South American mushroom growers to spawn substrate with their hand, without any mechanized help and in absence of care to avoid contamination. Thus, many of the contaminations that bags suffer with Trichoderma sp could occur during spawn phase. To learn more about the conditions that promote Trichoderma sp growth on lignocellulose substrates during spawning phase we designed a number of experiments in which substrates were treated with different methods commonly used to eliminate contaminations and then were inoculated with Trichodermasp previous to the inoculation with the mushroom spawn. To standardize the experiment, we firstly designed a method to inoculate the substrates with a spray of a suspension of conidia of Trichoderma sp. We used two mushroom species: P. ostreatus, widely worldwide cultivated (Lechner and Albertó, 2011) and Gymnopilus pampeanus a species which, at present, is being studied for mushroom production (Colavolpe and Albertó, 20122014). The latter can easily grow on sawdust of Populus and Eucalyptus but not on wheat straw. In our first experiment, results showed that sterilized substrates favor Trichoderma spgrowth (+). This result is in agreement with previous works (Velázquez-Cedeño et al., 2004Velázquez-Cedeño et al., 2006). It is really interesting to observe that T treatment on NS did not produce the growth of Trichoderma sp although it had a high concentration of conidia. It is supposed that S treatment due to high temperature and the cooking effect, released nutrients that benefited the green mold. It is also considered that the reduction of the natural microbial flora of the substrate by the sterilization action increases Trichoderma sp opportunities to colonize the substrate because of a lower presence of competitive micro flora which reduces the possibility of mycelial growth. Bacterial strains can inhibit the growth of Trichoderma sp by production of volatile organic compounds (Mackie and Whetley, 1999) or by releasing antibiotics (Nielsen et al., 2000). Species of bacteria belonging to genus Pseudomonas have been identified as antagonists of Trichoderma sp (Upadhyay et al., 1991Ellis et al., 2000). Velázquez-Cedeño et al. (2004) proved that the capacity of T. longibrachiatum to compete with P. ostreatus in dual cultures decreased in the presence of other micro-organisms in the substrates. The presence of total microflora increased the production of phenoloxidases by P. ostreatus despite a less abundant colonization of the substrate. The production of laccases has already been described as a response to environmental stress (Rayner et al., 1994Score et al., 1997Savoie et al., 2001). Velázquez-Cedeño et al. (2007) proved that Bacillus spp. and specifically Paenibacillus polymyxa from cultivation substrates are implicated in their selectivity by both inhibiting the growth of T. harzianum and stimulating defenses’ of the mushroom P. ostreatus through the induction of laccases.

We used four different substrates to test the growth of Trichoderma sp. Wheat straw which is commonly used for the production of Pleurotus specie in the region (Carabajal et al., 2012); wheat seed which is used for spawn production, Populus, frequently used for Pleurotus and Shiitake cultivation, and Eucalyptuswhich is also used for Shiitake (Pire et al., 2001) and Gymnopilus (Colavolpe and Albertó, 20122014). The growth of Trichoderma sp was high (+++) for all substrates for T+M treatment with S. No strong differences among substrates and treatments in the growth of Trichoderma sp were observed. Controls were free of contaminants. The source of contaminations in M treatments is unknown, it was probably present in the susbtrate and could survived due to a not enought sterlization time; for example sawdust (Table 3) in which longer steam treatments are needed.

Table 3

Growth of green mold disease after sterilization treatment*.

  Treatments   Substrates S NS     T T + M M C T T + M M C Wheat straw +++ (5) +++ (5) − (5) − (5) − (5) + (5) + (2) − (5) Wheat seed ++ (5) +++ (5) − (5) − (5) − (5) − (5) − (5) − (5) Populus sawdust ++ (5) +++ (5) + (2) − (5) − (5) − (5) − (5) − (5) Eucalyptus sawdust + (5) +++ (5) + (1) − (5) − (5) − (5) − (5) − (5)
*See Table 1 for experimental design.

T: Trichoderma sp (ICFC 767/12); T + M: Trichoderma + mushroom spawn (ICFC 153/99 for wheat straw and wheat seed; ICFC 548/03 for Populus and Eucalyptus sawdust), M: Mushroom spawn (ICFC 153/99 for wheat straw and wheat seed; ICFC 548/03 for Populus and Eucalyptus sawdust). S: Sterilized treatment, NS: non-sterilized treatment. C: Control (treatment without inoculation). (+): poor growth; (+ +): intermediate growth; (+ + +): abundant growth; (−): non-growth; the number between parentheses indicates number of replicates that obtained the result shown.

In a second experiment, the analysis of table 4 also reinforces this hypothesis. In fact, Trichoderma sp did not grow in 60 °C, 80 °C and NS treatments for T treatment. This is a relevant result if we take into account that the concentration of conidia of Thichoderma herein used is high and probably very difficult to find in natural substrates. We also used a high ratio of spawn (10%), trying to find a balance of forces between the concentration of conidia and the % of spawn. The lack of contamination of non-treated substrates may occur because of the microbiological quality of the substrate and also due to inability of Trichoderma sp to grow on “non-sterilized substrates” which is probably because of the poor assimilable nutrient availability. Trichoderma sp had difficulty in growing in M presence; the growth of M may affect some of the antagonistic bacteria present in NS and even thermoresistant ones present in 60 °C, or 80 °C. It is important to remark what happened with T+M treatments. In the case of 60 °C and 80 °C treatments, Trichoderma sp grew with Pleurotus ostreatus and with Gymnopilus pampeanus, in a smaller proportion than in S treatments but its growth was very clear. Controls were free of contaminants. We believe that there is an interaction between Trichoderma sp and the mushroom that favors green mold growth. This interaction could be due to the mushroom enzymes action. Enzymes of the mushroom were released to the media and produced an extracellular digestion of the substrate. The nutrients, now available for Trichoderma sp, could be partially absorbed by the fungus and used to colonize the substrate. It is interesting to point out that growth of Trichoderma sp was practically the same at 60 °C and 80 °C. So, if the mushroom grower uses an immersion in hot water treatment, 60 °C should be used to save energy and lower production costs. The immersion in hot water has some negative aspects: it uses a great amount of water, which could be a negative factor due to scarcity of this resource in some areas and it produces a reduction in yields due to the loss of nutrients extracted during heating by lixiviation (Jaramillo and Albertó, 2013). Additionally, the use of the alkaline method produces yields that can vary from 37 to 126% depending on the substrates (Contreras et al., 2004) and so is a preferable treatment method.

Table 4

Growth of green mold disease after immersion of substrates in hot water at 60, 80 °C or steam sterilization. Substrates were inoculated with a spray of Trichoderma (T) and then with spawn (M)*.

    Treatments     Substrates Mushroom 60 °C 80 °C S NS         T T + M M C T T + M M C T T + M M C T T + M M C Wheat straw 153/99 − (5) ++ (4) − (5) − (5) − (5) ++ (4) − (4) − (5) +++ (5) +++ (5) + (2) − (5) − (5) + (5) + (5) − (5) Populussawdust 153/99 − (5) + (5) − (5) − (5) − (5) ++ (5) − (5) − (5) +++ (4) +++ (5) + (1) − (5) − (5) + (4) − (5) − (5) Populussawdust 444/01 − (5) ++ (4) − (5) − (5) − (5) ++ (5) − (5) − (5) ++ (4) +++ (5) + (1) − (5) − (5) ++ (4) + (5) − (5)
*See Table 2 for experimental design.

T: Trichoderma sp (ICFC 767/12); M: Mushroom spawn (ICFC 153/99 or ICFC 444/01). S: Sterilized treatment, NS: non-sterilized treatment. C: Control (treatment without inoculation). (+): poor growth; (+ +): intermediate growth; (+ + +): abundant growth; (−): non-growth; the number between parentheses indicates number of replicates that obtained the result shown.

In the third experiment, we evaluated the growth of Trichoderma sp after the effect of the immersion of the substrate in alkalized water at different immersion times (Table 5). For T treatment, results showed that Trichoderma sp did not grow when period of treatment was 24 or 36 h. Trichoderma sp managed to develop for T+M treatment which means that Trichoderma sp could have been benefited by the enzymatic action of mushrooms in the co cultivation. The period of time in which the lower growth (+) was obtained was 36 h. Controls were free of contaminants. Some researchers suggested that adjusting pH to alkaline levels is a good means of inhibiting the growth of competitor fungi without seriously affecting the growth of P. ostreatus (Stölzer and Grabbe, 1991). Contreras et al. (2004) pointed out that no fungal contamination was found in the treated substrates; however certain group of bacteria like pseudomonads, bacilli and coliforms were detected. In this case bacteria action could have also helped to control Trichoderma sp development. For this experience, M treatments grew and colonized the substrate without any difficulty, which means that pH did not affect mushroom running. From this fact it may be concluded that adjusting pH by soaking in alkaline solution to alkaline levels is a good means of limiting the growth of green mold, and probably other fungi, without seriously affecting the growth of P. ostreatus. This confirms reports by Stölzer and Grabbe (1991) and Hernández et al. (Hernández et al., 2003), who suggested the use of alkaline pHs to cultivate this mushroom.

Table 5

Growth of green mold disease after substrate immersion in alkalinized water during different times. Substrates were inoculated with a spray of Trichoderma (T) and with Pleurotus ostreatus spawn (M).

Treatments Immersion in alkalinized water S     Duration of treatment                   Substrate Ut 5′ 12 h 24 h 36 h                   Wheat straw T T+M M C T T+M M C T T+M M C T T+M M C T T+M M C T T+M M C   −(5) +(4) +(2) −(5) +(4) ++(4) −(4) −(5) +(4) ++(4) −(4) −(5) −(5) ++(4) −(5) −(5) − (5) +(4) −(5) −(5) +++ (5) +++ (5) −(5) −(5)

T: Trichoderma sp. (ICFC 767/12), M: Mushroom spawn (ICFC 153/99). Ut: untreated; S: Sterilized treatment. C: Control (treatment without inoculation). (+): poor growth; (++): intermediate growth; (+++): abundant growth; (−): non-growth; the number between parentheses indicates number of replicates that obtained the result shown.

The results here obtained reinforce the hypothesis that mushroom cultivation treatments of the substrates could influence the growth of green mold disease. As a consequence, these treatments would also influence the contamination which may occur during spawning phase. Care for sanitary handling of spawn has to be considered by mushroom farmers. Also, inoculation of bags has to preferably be done in rooms instead of outdoors to avoid contaminations.

Factors that influence Trichoderma sp growth can be summarized as follows: i) the quality of the substrate (microbiological charge of contaminants) before treatment; ii) inability of Trichoderma sp to grow on non-sterilized substrates; iii) reduction of the natural microbial flora by the sterilization action which reduces competition for the substrate; iv) Co- cultivation with mushrooms which promotes Trichoderma sp growth probably by the release of nutrients easily assimilable (simple sugars); v) Immersion in an alkaline solution limits Trichoderma sp growth.

Conclusion

Mushroom cultivation disinfection treatments of lignocellulose substrates influence on the growth of green mold disease when contaminations occur during spawning phase. The immersion in hot water at 60 °C for 30 min and immersion in alkalinized water for 36 h are the recommended treatments to avoid contaminations with Trichoderma sp during spawning phase for the cultivation of xylophages species. Care for sanitary handling of spawn has also been considered by mushroom farmers to reduce contaminations. Further studies have to be carried out in order to determine the effects of the treatments on the release or immobilization of readily available nutrients (C and N) and the development of bacteria by focusing on those able to be antagonists of Trichoderma spp.

Acknowledgments

This research was made possible by the support of the Argentinian National Research Council (CONICET) and the National University of San Martín (UNSAM).

References
  • Ait-Lahsen H, Soler A, Rey M, de La Cruz J, Monte E, Llobell A. An antifungal exo-alpha-1, 3-glucanase (AGN 13.1) from the biocontrol fungus Trichoderma harzianum. Appl Environ Microbiol. 2001;67:5833–5839. [PMC free article] [PubMed] [Google Scholar]
    Ait-Lahsen H, Soler A, Rey M, de La Cruz J, Monte E, Llobell A (2001) An antifungal exo-alpha-1, 3-glucanase (AGN 13.1) from the biocontrol fungus Trichoderma harzianum. Appl Environ Microbiol 67:5833–5839. [
    PMC free article] [PubMed]
  • Carabajal M, Levin L, Albertó E, Lechner B. Effect of co-cultivation of two Pleurotus species on lignocellulolytic enzyme production and mushroom fructification. International Biodeterioration & Biodegradation. 2012;66(1):71–76. [Google Scholar]
    Carabajal M, Levin L, Albertó E, Lechner B (2012) Effect of co-cultivation of two Pleurotus species on lignocellulolytic enzyme production and mushroom fructification. International Biodeterioration & Biodegradation 66(1):71–76.
  • Castle A, Speranzini D, Rghei N, Alm G, Rinker D, Bissett J. Morphological and molecular identification of Trichoderma sp isolates on North American mushroom farms. Appl Environ Microbiol. 1998;64:133–137. [PMC free article] [PubMed] [Google Scholar]
    Castle A, Speranzini D, Rghei N, Alm G, Rinker D, Bissett J (1998) Morphological and molecular identification of Trichoderma sp isolates on North American mushroom farms. Appl Environ Microbiol 64:133–137. [
    PMC free article] [PubMed]
  • Colavolpe B, Albertó E. Anales del VII Simposio de Biotecnología Aplicada a la Agricultura.Universidad Paranaense; Umuarama: 2012. Avaliação da produção do cogumelo comestível Gymnopilus espectabilis var pampeanus em serragem de poplar e Eucalyptus. [Google Scholar]
    Colavolpe B, Albertó E (2012) Avaliação da produção do cogumelo comestível Gymnopilus espectabilis var pampeanus em serragem de poplar e Eucalyptus. Anales del VII Simposio de Biotecnología Aplicada a la Agricultura. Universidad Paranaense, Umuarama.
  • Colavolpe B, Albertó E. Cultivation requirements and substrate degradation of the edible mushroom Gymnopilus pampeanus- a novel species for mushroom cultivation. Scientia Horticulturae. 2014;180:161–166. [Google Scholar]
    Colavolpe, B. Albertó E (2014) Cultivation requirements and substrate degradation of the edible mushroom Gymnopilus pampeanus- a novel species for mushroom cultivation. Scientia Horticulturae 180:161–166.
  • Contreras EP, Sokolov M, Mejía G, Sánchez JE. Soaking of substrate in alkaline water as a pretreatment for the cultivation of Pleurotus ostreatus. Journal of Horticultural Science and Biotechnology. 2004;79(2):234–240. [Google Scholar]
    Contreras EP, Sokolov M, Mejía G, Sánchez JE (2004) Soaking of substrate in alkaline water as a pretreatment for the cultivation of Pleurotus ostreatus. Journal of Horticultural Science and Biotechnology 79(2):234–240.
  • Ellis RJ, Timms-Wilson TM, Bailey MJ. Identification of conserved traits in fluorescent pseudomonads with antifungal activity. Environ Microbiol. 2000;2:274–284. [PubMed] [Google Scholar]
    Ellis RJ, Timms-Wilson TM, Bailey MJ (2000) Identification of conserved traits in fluorescent pseudomonads with antifungal activity. Environ Microbiol 2:274–284. [
    PubMed]
  • Fletcher JT, White PF, Gaze RH. Mushrooms: Pest and Disease Control. Intercept Limited; Newcastle, England: 1986. p. 159. [Google Scholar]
    Fletcher JT, White PF, Gaze RH (1986) Mushrooms: Pest and Disease Control. Intercept Limited, Newcastle, England, 159 pp.
  • Geremia R, Goldman GH, Jacobs D, Ardiles W, Vila SB, Van Montagu M, Herrera-Estrella A. Molecular characterization of the proteinase-encoding gene, prb1, related to mycoparasitism by Trichoderma sp harxiunum. Mol Microbial. 1993;8:603–613. [PubMed] [Google Scholar]
    Geremia R, Goldman GH, Jacobs D, Ardiles W, Vila SB, Van Montagu M, Herrera-Estrella A (1993) Molecular characterization of the proteinase-encoding gene, prb1, related to mycoparasitism by Trichoderma sp harxiunum. Mol Microbial 8:603–613. [
    PubMed]
  • Hatvani L, Antal Z, Manczinger L, Szekeres A, Druzhinina IS, Kubicek CP, Nagy A, Nagy E, Vágvölgyi C, Kredics L. Green mold diseases of Agaricus and Pleurotus spp. are caused by related but phylogenetically different Trichoderma sp species. Phytopathology. 2007;97:532–537. [PubMed] [Google Scholar]
    Hatvani L, Antal Z, Manczinger L, Szekeres A, Druzhinina IS, Kubicek CP, Nagy A, Nagy E, Vágvölgyi C, Kredics L (2007) Green mold diseases of Agaricus and Pleurotus spp. are caused by related but phylogenetically different Trichoderma sp species. Phytopathology 97:532–537. [
    PubMed]
  • Hermosa MR, Grondona I, Monte E. Isolation of Trichoderma sp harzianum Th2 from commercial mushroom compost in Spain. Plant Dis. 1999;83:591. [Google Scholar]
    Hermosa MR, Grondona I, Monte E (1999) Isolation of Trichoderma sp harzianum Th2 from commercial mushroom compost in Spain. Plant Dis 83:591.
  • Hernández D, Sánchez JE, Yamasaki K. Composting, a simple procedure for preparing substrate for cultivation of Pleurotus ostreatus. Bioresource Technology. 2003;90(2):145–50. [PubMed] [Google Scholar]
    Hernández D, Sánchez JE, Yamasaki, K (2003) Composting, a simple procedure for preparing substrate for cultivation of Pleurotus ostreatus. Bioresource Technology 90(2):145–50. [
    PubMed]
  • Jandaik S, Guleria DS. Yield loss in Agaricus bisporus due to Trichoderma sp infection. Mushroom Res. 1999;8:43–46. [Google Scholar]
    Jandaik S, Guleria DS (1999) Yield loss in Agaricus bisporus due to Trichoderma sp infection. Mushroom Res 8:43–46.
  • Jaramillo S, Albertó E. Heat treatment of wheat straw by immersion in hot water decreases mushroom yield in Pleurotus ostreatus. Rev Iberoam Micol. 2013;30(2):125–129. [PubMed] [Google Scholar]
    Jaramillo S, Albertó E (2013) Heat treatment of wheat straw by immersion in hot water decreases mushroom yield in Pleurotus ostreatus. Rev Iberoam Micol 30(2):125–129. [
    PubMed]
  • Komon-Zelazowska M, Bissett J, Zafari D, Hatvani L, Manczinger L, Woo S, Lorito M, Kredics L, Kubicek CP, Druzhinina IS. Genetically closely related but phenotypically divergent Trichoderma spspecies causes green mold diseases in oyster mushroom farms worldwide. Applied & Environmental Microbiology. 2007;73(22):7415–7426. [PMC free article] [PubMed] [Google Scholar]
    Komon-Zelazowska M, Bissett J, Zafari D, Hatvani L, Manczinger L, Woo S, Lorito M, Kredics L, Kubicek CP, Druzhinina IS (2007) Genetically closely related but phenotypically divergent Trichoderma sp species causes green mold diseases in oyster mushroom farms worldwide. Applied & Environmental Microbiology 73(22):7415–7426. [
    PMC free article] [PubMed]
  • Krupke OA, Castle AJ, Rinker DL. The North American mushrooms competitor, Trichoderma sp aggressivum f. aggressivum, produces antifungal compounds in mushroom compost that inhibit mycelial growth of the commercial mushroom Agaricus bisporus. Mycological Research. 2003;107(12):1467–1475. [PubMed] [Google Scholar]
    Krupke OA, Castle AJ, Rinker DL (2003) The North American mushrooms competitor, Trichoderma sp aggressivum f. aggressivum, produces antifungal compounds in mushroom compost that inhibit mycelial growth of the commercial mushroom Agaricus bisporus. Mycological Research 107(12):1467–1475. [
    PubMed]
  • Lechner BE, Albertó E. Search for new naturally occurring strains of Pleurotus to improve yields. P. albidus as a novel proposed species for mushroom production. Rev Iberoam Micol. 2011;28:148–154. [PubMed] [Google Scholar]
    Lechner BE, Albertó E (2011) Search for new naturally occurring strains of Pleurotus to improve yields. P. albidus as a novel proposed species for mushroom production. Rev Iberoam Micol 28:148–154. [
    PubMed]
  • Mackie AE, Whetley RE. Effects and incidence of volatile organic compound interactions between soil bacterial and fungal isolates. Soil Biol Biochem. 1999;3:375–385. [Google Scholar]
    Mackie AE, Whetley RE (1999) Effects and incidence of volatile organic compound interactions between soil bacterial and fungal isolates. Soil Biol Biochem 3:375–385.
  • Mamoun ML, Savoie JM, Olivier JM. Interactions between the pathogen Trichoderma sp harzianumTh2 and Agaricus bisporus in mushroom compost. Mycologia. 2000;92:233–240. [Google Scholar]
    Mamoun ML, Savoie JM, Olivier JM (2000) Interactions between the pathogen Trichoderma sp harzianum Th2 and Agaricus bisporus in mushroom compost. Mycologia 92:233–240.
  • Nielsen TH, Thrane C, Christophersen C, Anthoni U, Sorensen J. Structure, production characteristics and fungal antagonism of tensin - A new antifungal cyclic lipopeptide from Pseudomonas fluorescens strain 96.578. J Appl Microbiol. 2000;89:992–1001. [PubMed] [Google Scholar]
    Nielsen TH, Thrane C, Christophersen C, Anthoni U, Sorensen J (2000) Structure, production characteristics and fungal antagonism of tensin - A new antifungal cyclic lipopeptide from Pseudomonas fluorescens strain 96.578. J Appl Microbiol 89:992–1001. [
    PubMed]
  • Pieckenstain F, Mercuri O, Albertó E. Mevinolin in naturally occurring specimens of Pleurotus cornucopiae. Micol Neotrop Aplicada. 1999;12:1–7. [Google Scholar]
    Pieckenstain F, Mercuri O, Albertó E (1999) Mevinolin in naturally occurring specimens of Pleurotus cornucopiae. Micol Neotrop Aplicada 12:1–7.
  • Pire G, Wright JE, Albertó E. Cultivation of shiitake using sawdust of widely available local woods in Argentina. Mic Int Aplic. 2001;13(2):1–5. [Google Scholar]
    Pire G, Wright JE, Albertó E (2001) Cultivation of shiitake using sawdust of widely available local woods in Argentina. Mic Int Aplic 13(2):1–5.
  • Rayner ADM, Griffith GS, Wildman HG. Induction of metabolic and morphogenetic changes during mycelial interactions among species of higher fungi. Biochem Soc Trans. 1994;22:389–394.[PubMed] [Google Scholar]
    Rayner ADM, Griffith GS, Wildman HG (1994) Induction of metabolic and morphogenetic changes during mycelial interactions among species of higher fungi. Biochem Soc Trans 22:389–394. [
    PubMed]
  • Savoie JM, Mata G, Mamoun M. Variability in brown line formation and intracellular laccase production during interaction between white-rot basidiomycetes and Trichoderma sp harzianumbiotype. Mycología. 2001;93:243–248. [Google Scholar]
    Savoie JM, Mata G, Mamoun M (2001) Variability in brown line formation and intracellular laccase production during interaction between white-rot basidiomycetes and Trichoderma sp harzianumbiotype. Mycología 93:243–248.
  • Score AJ, Palfreyman JW, White NA. Extracellular phenoloxidase and peroxidase enzyme production during interspecific fungal interaction. Int Biodeterior Biodegrad. 1997;39:225–233.[Google Scholar]
    Score AJ, Palfreyman JW, White NA (1997) Extracellular phenoloxidase and peroxidase enzyme production during interspecific fungal interaction. Int Biodeterior Biodegrad 39:225–233.
  • Sharma SR, Vijay B. Yield loss in Pleurotus ostreatus spp. caused by Trichoderma sp viride. Mushroom Res. 1996;5:19–22. [Google Scholar]
    Sharma SR, Vijay B (1996) Yield loss in Pleurotus ostreatus spp. caused by Trichoderma sp viride. Mushroom Res 5:19–22.
  • Sivan A, Chet I. Degradation of fungal cell walls by lytic enzymes from Trichoderma sp harzianum. J Gen Microbiol. 1989;135:675–682. [Google Scholar]
    Sivan A, Chet I (1989) Degradation of fungal cell walls by lytic enzymes from Trichoderma sp harzianum. J Gen Microbiol 135:675–682.
  • Stölzer S, Grabbe K. Mechanisms of substrate selectivity in the cultivation of edible fungi. In: Maher MJ, editor. Mushroom Science 13 Science and Cultivation of Edible Fungi. Balkema; Rotterdam: 1991. pp. 141–146. [Google Scholar]
    Stölzer S, Grabbe K (1991) Mechanisms of substrate selectivity in the cultivation of edible fungi. In: Maher, MJ (ed) Mushroom Science 13. Science and Cultivation of Edible Fungi. Balkema, Rotterdam, pp 141–146.
  • Tokimoto K, Komatsu M. Effect of carbon and nitrogen sources in media on the hyphal interference between Lentinus edodes and some species of Trichoderma sp. Annals of the Phytopathological Society of Japon. 1979;45:261–264. [Google Scholar]
    Tokimoto K, Komatsu M (1979) Effect of carbon and nitrogen sources in media on the hyphal interference between Lentinus edodes and some species of Trichoderma sp. Annals of the Phytopathological Society of Japon 45:261–264.
  • Upadhyay RS, Visintin L, Jayaswal K. Environmental factors affecting the antagonism of Pseudomonas cepacia against Trichoderma sp viride. Can J Microbiol. 1991;37:880–884. [PubMed] [Google Scholar]
    Upadhyay RS, Visintin L, Jayaswal K (1991) Environmental factors affecting the antagonism of Pseudomonas cepacia against Trichoderma sp viride. Can J Microbiol 37:880–884. [
    PubMed]
  • Velázquez-Cedeño M, Farnet AM, Ferre E, Savoie JM. Variation of lignocellulosic activities in dual cultures of Pleurotus ostreatus and Trichoderma sp longibrachiatum on unsterilized wheat straw. Mycologia. 2004;96:712–719. [PubMed] [Google Scholar]
    Velázquez-Cedeño M, Farnet AM, Ferre E, Savoie JM (2004) Variation of lignocellulosic activities in dual cultures of Pleurotus ostreatus and Trichoderma sp longibrachiatum on unsterilized wheat straw. Mycologia 96:712–719. [
    PubMed]
  • Velázquez-Cedeño MA, Farnet AM, Mata G, Savoie JM. Estudio preliminar de la microflora bacteriana termo tolerante de la pulpa de café y la paja de trigo con potencial de inhibición contra Trichoderma sp viride en el cultivo de Pleurotus. Rev Mex Micol. 2006;22:33–39. [Google Scholar]
    Velázquez-Cedeño MA, Farnet AM, Mata G, Savoie JM (2006) Estudio preliminar de la microflora bacteriana termo tolerante de la pulpa de café y la paja de trigo con potencial de inhibición contra Trichoderma sp viride en el cultivo de Pleurotus. Rev Mex Micol 22:33–39.
  • Velazquez-Cedeno M, Farnet AM, Mata G, Savoie JM. Role of Bacillus spp. in antagonism between Pleurotus ostreatus and Trichoderma harzianum in heat-treated wheat-straw substrates. Bioresource Technology. 2007;99:6966–6973. [PubMed] [Google Scholar]
    Velazquez-Cedeno M, Farnet AM, Mata G and Savoie JM (2007). Role of Bacillus spp. in antagonism between Pleurotus ostreatus and Trichoderma harzianum in heat-treated wheat-straw substrates. Bioresource Technology 99:6966–6973. [
    PubMed]

Edited by WolfWhiz, 14 April 2019 - 05:16 AM.

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#2 Billcoz

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Posted 14 April 2019 - 08:43 AM

Yeah, trich sucks. I have 2 upstairs rooms, one has my weed grow tent and the other is a small(8x10) room/walk in closet with no door, that's where my mush grow is. they're kind of around the corner from each other. I have some plants in the tent that I have re-vegged a couple times now so the coir in the pots is pretty old, and has visible trich around the plant base. Luckily I haven't had any  of that forest green shit get over into the mush grow, but I did have a spent sub in a tub I was gonna toss out sitting over in the room with the tent, sitting there for like 5-6 days before I tossed it(I'm lazy), and when I did I saw it had some dark green on the sub. I immediately got rid of it and cleaned the area and tub. I keep the space between the rooms clean and have had no problems in the mush-room yet, hopefully I never do. I toss sub after only 3 flushes, sometimes 2 if I want to make room for new strains, so that helps keep trich down, as old subs have been exposed and contams can build up on it. Thanks for all that info dude, it's a long ass post.


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#3 Billcoz

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Posted 14 April 2019 - 08:55 AM

This is great info, it's very specific about sterilizing subs etc, though I still sometimes spawn to sterilized coir, I think the lack of nutrients in coir is not conducive to contam growth, at least I haven't seen it.


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#4 bezevo

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Posted 14 April 2019 - 07:09 PM

do you mean sterilize or pasteurize ?

 

seems most common held belief is your more likly to have contams on a sterilized sub vers a pasteurized sub with beneficial  bacteria .

 

hummm well thats what most info i read says .


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#5 raymycoto

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Posted 15 April 2019 - 02:44 PM

I'm amazed at how resistant to trich subs are once they really get colonized. Even really old and burned out and ready to toss, I never see trich on the old substrate. Although I do see an increasing problem with trich in grain jars recently and with some of my early sub colonization.

Perhaps an old sub is just so colonized by the desired mushroom and there is no way that trich can take hold. But I wonder if there is some sort of humoral factor that helps to defend the colonized sub against infection. If so, could one take a bit of the fluid from the sub and inoculate a new sub with it for trich prevention? That water would be really dirty and no telling what is really growing in it.

I got a ton of experiments going and on the wish list but that's one of them. Perhaps prepare some of that nasty trich liquid culture (would hate to actually cultivate trich!) then make a couple of small bins. Inoculate some sub with spawn and spray or somehow hit one of them with some old substrate water.

Well, I do happen to have a large jar grain that I just identified as having a few spots of trich. Sadly, I know that it will probably propogate this to a new substrate before the myc can take over. Perhaps I'll divide it in two and do that experiment with that jar.


Edited by raymycoto, 15 April 2019 - 02:46 PM.

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#6 Billcoz

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Posted 15 April 2019 - 05:02 PM

do you mean sterilize or pasteurize ?

 

seems most common held belief is your more likly to have contams on a sterilized sub vers a pasteurized sub with beneficial  bacteria .

 

hummm well thats what most info i read says .

What I meant was that it does not matter as much with coir as with manure, sterilized coir does not seem to contam any more that just pasteurized coir, but pasteurizing without sterilizing is recommended for shits.

 

The post talks how pasteurizing substrate kills off "bad" bacteria, it lists some "good" species of bacteria that sterilizing kills but pasteurizing does not kill and are beneficial to stop contams from colonizing.


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#7 Billcoz

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Posted 15 April 2019 - 05:07 PM

I'm amazed at how resistant to trich subs are once they really get colonized. Even really old and burned out and ready to toss, I never see trich on the old substrate. Although I do see an increasing problem with trich in grain jars recently and with some of my early sub colonization.

Perhaps an old sub is just so colonized by the desired mushroom and there is no way that trich can take hold. But I wonder if there is some sort of humoral factor that helps to defend the colonized sub against infection. If so, could one take a bit of the fluid from the sub and inoculate a new sub with it for trich prevention? That water would be really dirty and no telling what is really growing in it.

I got a ton of experiments going and on the wish list but that's one of them. Perhaps prepare some of that nasty trich liquid culture (would hate to actually cultivate trich!) then make a couple of small bins. Inoculate some sub with spawn and spray or somehow hit one of them with some old substrate water.

Well, I do happen to have a large jar grain that I just identified as having a few spots of trich. Sadly, I know that it will probably propogate this to a new substrate before the myc can take over. Perhaps I'll divide it in two and do that experiment with that jar.

Isaid this in an earlier post but I did have some trich on a sub that was in a tub near my weed grow tent, which has trich in it for sure, I see it on the coir in the plant pots in the tent. It was an old sub I had gotten 5 flushes from, I had left it there planning to toss it out but I got lazy. When I finally did go to toss it(after a week or more) I noticed dark green patches on the sub. I just keep the mush grow area clean, and I spray lysol around(careful not to get any on myc, it causes mutations). I've never seen any green in my mush grow room.


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#8 sandman

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Posted Yesterday, 07:03 AM

You wont hardly ever see trich before at least 2 flushes when you use agar and properly sterilized grains and properly pasteurized substrates with a good sterile technique. If you are seeing a lot of trich that just means you need to work on one of those areas. 


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#9 WolfWhiz

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Posted Yesterday, 01:01 PM

hummm well thats what most info i read says .

Ahh, is this the new PC term for the old "AFOAF:  :biggrin:  :biggrin:  :biggrin:    :tongue:  :tongue:  :tongue:



#10 PJammer24

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Posted Today, 11:00 AM

Trich? What is this Trich you speak of? The winter months have rocked... I will likely be reminded of what Trich is all about this summer, but fingers crossed...

 

a few HEPA filters strategically placed seems to help with the old Trichoderma... 



#11 raymycoto

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Posted Today, 01:50 PM

Do you have an opinion on a temp range that promotes trich over the desired mycelium? I have mainly played with cubes but I now have 4 other edibles in the pipeline so I want to protect them from the dreaded problem.

 

I have an incubation are and it is at about 80F. Seems a bit of trich has been popping up. I'm not blaming the temp nor the incubation area. Just thinking about what is the optimal temp for cube (for ex) and the optimal for trich. And if a shift in temp of some direction may help.

 

Incidentally, I've been playing with a continuous grain to grain idea of keeping a starter of a colonized grain then doing a 1:1 G-G mix with sterile grain and so on to ramp up the colonized grain. Then when I get to a well colonized and good quantity, put into substrate and keep some to repeat the G-G process. Well I realize that senescence may be an issue but I could not resist trying this.

 

Problem with the above is this - suppose you have a tiny speck of trich. Unlike your desired myc, it sporulates right away and that is how you can even see it! So this tiny spec then colonizes the next grain jar with tons of spores. After about 3 transfers, it's won the battle because it is like mixing your myc with a spore syringe of trich spores. At least that is my theory. Thus, the perpetual Grain-to-Grain - idea - BUSTED - ha! Well it was worth trying.

 

Maybe add some peroxide in my G-G transfers? Probably not a solution. I've played with peroxide. It does inhibit spores but it also inhibits myc for the time required for it to develop the peroxidase to continue to grow.


Edited by raymycoto, Today, 01:51 PM.


#12 jkdeth

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Posted Today, 02:08 PM

Trich likes hot. Over 75 degrees. Cooler temperatures won't stop it, but will slow it. It also likes acidic ph. Bump up to alkaline 7.5 or above.

Grain masters are common. If I recall correctly 4th expansion should be the last. If that were a goal, really needs to be flawless. Grain started from clean agar. MS inoculation shouldn't even be considered. Its always a race with ms inoculation, obviously the myc can win, but the long its allowed to grow the more chance of trich "coming from behind" and overtaking the mycelium. Its not uncommon to see g2g fail from ms, even if the first grain jar seems fine.




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