Forest farming of shiitake mushrooms: An integrated evaluation of management practices

Two outdoor shiitake (Lentinula edodes) cultivation experiments, established in Missouri USA in 1999 and 2000, produced mushrooms in 2000–2005. We examined shiitake production in response to substrate species, inoculum form, inoculum strain, and inoculation timing, using total mushroom weight per log as the primary response variable with log characteristics as covariates. The significantly greater mushroom weight produced by sugar maple logs compared with white or northern red oak was attributable to the higher proportion of undiscolored wood volume in the maple logs, rather than to bark thickness or log diameter. The “wide temperature range” shiitake strain produced significantly greater yield compared with the “warm” or “cold” weather strains. Both the wide-range and warm-weather strains were stimulated to fruit by significant rain events, while the cold-weather strain was responsive to temperature. Inoculation with sawdust spawn gave significantly greater yield than colonized wooden dowels or pre-packaged “thimble” plug inoculum. The second and third full years following inoculation were the most productive.     

Effect of liquid mycelial culture used as a spawn on sawdust cultivation of shiitake (Lentinula edodes)

Mycelium of shiitake (Lentinula edodes) was cultured continuously in liquid medium. The liquid culture was carried out for the production of liquid spawn in the cultivation of this mushroom on synthetic sawdust substrate, and its performance was compared with that of the solid spawn. The initial colonization in culture bags was faster with the solid spawn than with the liquid spawn, but after this stage CO₂ production was higher with the liquid spawn than with the solid spawn. For harvesting sufficient amount of good quality mushrooms, 120 d of incubation in bags was needed with the solid spawn, but this was reduced to 90 d for the sawdust blocks using liquid spawn of less than 50 d old. If continuous culture of the liquid spawn was prolonged over 50 d, immature fruit-bodies or their initials formed during the period of bag incubation. The solid subcultures of the liquid spawns retained the fruiting characteristics acquired in the liquid culture. Liquid culture could be a useful tool for breeding of mushrooms.     

Spatial and temporal patterns of morel fruiting

The biotic and abiotic factors conditioning morel fruit body production are incompletely known. We examined spatial and temporal patterns of Morchella esculenta fruiting over five years in a wooded site in Missouri, USA. Fruiting onset was inversely correlated with spring air and soil temperatures, whereas abundance was positively correlated with rain events (>10 mm) during the 30 d preceding fruiting. The two years with the greatest fruiting had the shortest fruiting seasons (6–7 d). Fruiting season length was positively correlated with soil warming, suggesting that a narrow range of optimum soil temperatures favour the explosive production of fruit bodies. All woody stems of at least 1 cm diam were mapped and stem diameter and crown condition were noted. Morel fruit bodies were significantly closer to stems of Carya spp., Tilia americana and Ulmus americana than predicted by the frequencies of these woody species or their contribution to the total basal area on the site. Although intra-annual clustering of fruit bodies was often observed, inter-annual clustering was not. The spatial pattern of M. esculenta fruiting appears to be associated with vegetation pattern, whereas the onset and abundance of fruiting are determined by the interaction of spring temperatures with availability of supporting precipitation.     

Biological and inherited characteristics of a newly identified <Emphasis Type="Italic">Lentinula edodes</Emphasis> strain capable of forming a fruiting body without a shift to low temperature

A strain of the basidiomycete Lentinula edodes (Shiitake) was newly identified from the mushroom library of Mori Sangyo Co., Ltd., Japan. This strain, named MIL-LEW-M13-1, is capable of forming the fruiting body on sawdust-based medium without a reduction in temperature. Mating experiments with a monokaryotic mycelium of L. edodes strain that does require low temperature for fruiting–body formation suggest that the unique property of the MIL-LEW-M13-1 strain is a dominant trait that can be inherited by its progeny in a nucleus-dependent manner.     

Strain typing of shiitake (<Emphasis Type="Italic">Lentinula edodes</Emphasis>) cultivars by AFLP analysis,focusing on a heat-dried fruiting body

To validate strain typing by amplified fragment length polymorphism (AFLP) analysis in shiitake (Lentinula edodes) cultivars, the reproducibility of AFLP markers with DNA extracted from the heat-dried fruiting body was evaluated. DNAs were extracted from three different portions of the heat-dried fruiting body – the stipe, pileus, and gill – and AFLP analysis of all parts was carried out using two combinations of selected amplification primer pairs. AFLP profiles of DNA from the gill tissue of heat-dried fruiting body were almost identical to those of cultured mycelia in the same strains, although it was difficult to detect reproducible AFLP profiles from stipe and pileus DNA. These results indicated that AFLP analysis would be applicable for strain typing with heat-dried fruiting bodies of L. edodes by using the DNA extracted from gills.Contribution No. 364 of the Tottori Mycological Institute     

Comparative studies on the diversity of the edible mushroom Pleurotus eryngii: ITS sequence analysis, RAPD fingerprinting, and physiological characteristics

Verification of Pleurotus eryngii strains was assessed using ITS sequence analysis and RAPD fingerprinting. Sequence analysis of the ITS1–5.8S rDNA–ITS2 region of 24 strains of Pleurotus sp., which consisted of 22 strains of P. eryngii and the control strains P. ostreatus and P. ferulae, demonstrated that the DNA regions share mostly 99 % sequence identity, indicating that sequence-based analysis is not applicable for the verification of closely related mushroom strains. To verify the mushroom strains using RAPD, we amplified DNA fragments from the total cellular DNA of 24 mushroom strains with 18 different random primers, yielding 538 distinct DNA fragments ranging from 200–4000 bp. Analysis of the DNA fragment pattern showed that the 22 P. eryngii strains were clearly distinguished from the control strains P. ostreatus and P. ferulae, and could be categorized into five subgroups. Subsequent physiological studies on the development of fruiting bodies demonstrated the close correlation of the RAPD-based grouping with the phenotypical characteristics of mushroom fruiting bodies.     

Characterization of vitamin B12 compounds in the fruiting bodies of shiitake mushroom (Lentinula edodes) and bed logs after fruiting of the mushroom

This study determined the vitamin B₁₂ content in commercially available dried fruiting bodies of shiitake mushroom, Lentinula edodes. The vitamin B₁₂ contents in dried donko-type fruiting bodies with closed caps (5.61 ± 3.90 μg/100 g dry weight), did not significantly differ from those of dried koushin-type fruiting bodies with open caps (4.23 ± 2.42 μg/100 g dry weight). The bed logs after fruiting of the mushroom also contained the vitamin B₁₂ levels similar to that in the dried shiitake fruiting bodies. To determine whether the dried shiitake fruiting bodies and their bed logs contained vitamin B₁₂ or other corrinoid compounds that are inactive in humans, we purified corrinoid compounds using an immunoaffinity column and identified vitamin B₁₂ using vitamin B₁₂-dependent Escherichia coli 215 bioautograms and liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) chromatograms. Dried shiitake fruiting bodies rarely contained an unnatural corrinoid vitamin B₁₂[c-lactone] that is inactive in humans. Given that shiitake mushroom lacks the ability to synthesize vitamin B₁₂ de novo, the vitamin B₁₂ found in dried shiitake fruiting bodies must have been derived from the bed logs.     

Adapting substrate formulas used for shiitake for production of brown Agaricus bisporus

A pasteurized, non-composted substrate (basal mixture) consisting of oak sawdust (28%), millet (29%), rye (8%), peat (8%), alfalfa meal (4%), soybean flour (4%), wheat bran (9%), and CaCO3 (10%) was adapted from shiitake culture to produce the common cultivated mushroom (brown; portabello), Agaricus bisporus. Percentage biological efficiency (ratio of fresh mushroom harvested/oven-dry substrate weight, %BE) ranged from a low of 30.1% (when wheat straw was substituted for sawdust) to 77.1% for the basal mixture. Special, high gas-exchange bags were required to optimize mycelial growth during spawn run. Our formula may allow specialty mushroom growers to produce portabello mushrooms on a modified, pasteurized (110 degrees C for 20 min) substrate commonly used for shiitake production without the added expense of compost preparation.     

The annual reproductive cycle of the freshwater mussel Dreissena polymorpha Pallas in lakes

Summary The annual development of the gonads of Dreissena polymorpha was studied at three sampling sites in two lakes over 3 and 1 1/2 years, respectively. A resting stage occurred after the last spawning in summer/autumn. Oogenesis (accompanied by multiplying segmentation of the oogonia and early growth processes of its oocytes) restarted in specimens at least 1 year old at low temperatures (below 10° C) during winter and early spring. At one location (Fühlinger See) the onset of the spawning season was correlated with an increase of water temperatures above 12° C. At 2 m depth, two main spawning periods in May and August were normally recognized, the first at temperatures of 12–16° C, the second at 16–21° C. It was clearly demonstrated for the first time in Dreissena polymorpha that the oocytes became mature in successive cohorts within one gonad. A female mussel may spawn several times during the reproductive season. At 9 m depth, the onset of spawning also started at about 12° C; this occurred in late summer, with two spawning periods within 1 month at a temperature range of 12–16° C. At another location (Heider Bergsee) the size of the gonads and the oocytes was reduced during April of both years studied, when food supply was low simultaneously with rapidly rising water temperatures in this shallow lake. There was no spawning period during spring. The major spawning period was delayed until July (temperatures 19–22°C). This shows (1) the synchronizing influence of low winter temperatures on the annual reproductive cycle and (2) a temperature threshold of at least 12° C for the start of the spawning processes. The results are discussed with regard to the geographical limits of further spread of Dreissena polymorpha.     

Growth and habitat separation in eight cohorts of three species of cyprinids in a subalpine lake

Synopsis Distribution and growth of the embryos, larvae and juveniles of Rutilus rutilus (roach), Scardinius erythrophthalmus (rudd) and Leuciscus cephalus (chub) from an oligotrophic subalpine lake in Tyrol, Austria, were studied during the first three to four months after hatching. R. rutilus was the first to spawn, a single cohort hatching around May 23rd. Four cohorts of S. erythrophthalmus hatched between June 19 and August 1. Three cohorts of L. cephalus hatched between July 3 and 25. The length/weight relationship of all species changed at a length of approximately 15–16 mm. R. rutilus, hatching at the lowest temperature, also showed the lowest growth rate during early life (maximum 10.4 per cent fresh body weight day^–1). In the other two species relative growth rates up to 20% day^–1) were measured. Rudd and chub remained in the shallow littoral during the whole period of observation, whereas roach left the littoral a few weeks after hatching and migrated into deeper water. A subtle shift in vertical distribution was observed for the first cohort of rudd which moved into slightly deeper water when the second cohort made its appearance.To whom correspondence should be addressed     

Studies on mixed mass cultivation of Anabaena spp. (nitrogen-fixing blue-green algae, cyanobacteria) on a large scale

Studies on mixed mass cultivation of Anabaena spp. on a large scale (5170m²) were conducted continuously for 3 years. Under the continental monsoon climate in northern subtropics (30°N, 115°E), 7–11 g dry weight m⁻² day⁻¹ of microalgal biomass on average was harvested in simple plastic greenhouses in the effective growth days during the warmer seasons. The maximum productivity was 22 g m⁻² day⁻¹ in the middle of summer. Observations on the productive properties of strains of Anabaena spp. indicated that they were different from and could compensate for each other in their productivities and adaptations to the seasonal changes. With different lining materials (PVC sheets, concrete, sand and soil) in the culture ponds, no significant variation of productivity was found, but bubbling with biogas in the middle of the day and the application of some growth regulating substances (2,4-D, NaHSO₃ and extracts of oyster mushroom spawn) was able to improve the production. The cost of microalgal biomass in this way was around 0·75–1·0 US dollar(s) per kilogram.     

Host feeding,damage and control of the mushroom pest,Brennandania lambi (Acari: Pygmephoroidea) in China

The pygmephoroid mite, Brennandania lambi (Krczal), is the most serious mite pest of mushroom production in Shanghai. China. This mite could not develop and reproduce on the mycelia of Lentinus edodes, Flammulina velutipes, Pleurotus ostreatus or Pleutotus sajor-caju, but 12–20% of the females survived 15 to 31 days on these hosts, whereas all mites died after 8 days in absence of food. When fed the mycelia of Auricularia polytricha, B. lambi adults could become gravid and reproduce but active larvae failed to develop to the next stage. B. lambi developed and reproduced on the mycelia of Auricularia auricula, Hericium erinaceus, Tremella fuciformis and Agaricus bisporus. Mobile stages of B. lambi damage A. bisporus by feeding on its mycelia. Field surveys of mite damage during 1983–1989 in the suburbs of Shanghai revealed that contaminated spawn was the major source of the mite problem and losses of the crop averaged between 10% and 20%. Experiments on the effects of temperature on mite survival indicate that exposure of mites at 50° C for 1 hour or-10°C for 24 hours killed all stages of B. lambi. Thus, heating treatment during well-executed phase II composting and freezing of mushroom spawn at-10° C for 24 hours should eliminate or at least reduce the damage to mushrooms by this pest.     

Mutants of Myxococcus xanthus insensitive to glycerol-induced myxospore formation

Mutants of Myxococcus xanthus FB_t unable to form myxospores in response to 0.5 M glycerol arise spontaneously with a frequency of 1–3×10^–5. These mutants are designated glc. Ultraviolet mutagenesis increases the frequency to a maximum of 7% of the survivors. The reversion frequency following ultraviolet irradiation of spontaneous glc mutants is less than 10^–3. Of four glc mutants examined, none form myxospores in response to the alternative inducers, ethylene glycol and dimethyl sulphoxide. One glc mutant is induced by 1.5 M glycerol; strain FB_t responds to this glycerol concentration with low efficiency myxospore formation. Strain FB_t and glc mutants all produce myxospores with low efficiency in response to phenyl ethanol. Of 117 glc mutants tested, 109 form fruiting bodies containing mature myxospores; thus, mutations to the glc phenotype do not normally block myxospore formation within the fruiting cycle of the organism.     

Recycling spent larval food of Corcyra cephalonica Stainton for preparing spawn and sporophore of Pleurotus sajor-caju (Fr.) Singer

Large scale production of the rice moth, Corcyra cephalonica Stainton in pearl millet grain medium leads to a huge accumulation of spent larval medium in commercial insectaries. We attempted bioconversion of spent larval medium of C. cephalonica (CLM) for cultivation of the mushroom Pleurotus sajor-caju (Fr.) Singer, to increase the usage of these residues. Maximum efficiency limits of CLM for spawn run, sporophore cropping and as bed substrate were assessed with varying combinations of sorghum and rice straw. Sorghum grains and rice straw were the best substrates for spawn run and sporophore yield respectively. Having been crushed, macerated, heated and sterilized, CLM could also become a suitable substrate along with sorghum or rice straw. Sorghum and CLM at 16.7% + 83.3% and 33.3% + 66.7% combinations were very effective in supporting mycelial growth and quicker colonization of fungus, and mother spawn yield. The spawn that was obtained from these combinations yielded higher sporophore as well. The fungus did not rapidly colonize on other combinations (50% + 50%, 66.7% + 33.3% and 83.3% + 16.7%), and was completely unable to grow on CLM 100%. Combination of rice straw and CLM at 75% + 25% and 50% + 50% as bed substrate contributed higher sporophore yield. Analysis of the substrates indicated variation in their chemical and mineral composition, but they were good sources of N, P and Ca. The prospects of exploring CLM for the mushroom cultivation are discussed.     

Spatial aspects of reproduction in buffalo sculpin,Enophrys hison

Synopsis The spatial distribution and parental care of demersal egg masses of buffalo sculpin (Enophrys bison) were studied at 11 areas in Puget Sound, Washington, during 1972–1973. Adult buffalo sculpin inhabited rock and rock-sand habitats at depths from the intertidal to minus 20 m throughout the year, but 84% of all spawn clusters were found at depths between –1 m and +1 m within localized rocky areas. Areas used for spawning were exposed to strong tidal currents and other water movements. A single male fish guarded and fanned spawn, but eggs were temporarily deserted when less than about 1 m of water covered the spawning site. The cyclical nature of male desertion, behavior of spawn predators, and spawn mortality due to physical factors while emergent suggest several possible advantages to low intertidal spawning. These advantages include spatial and temporal escape from marine predators by embryos, time for the tending parent to forage, and optimal physical conditions for embryonic development.     

Biodegradation of gossypol by the white oyster mushroom,Pleurotus florida,during culturing on rice straw growth substrate,supplemented with cottonseed powder

The capacity of the white oyster mushroom, Pleurotus florida to biodegrade gossypol was studied, when grown on rice straw supplemented with cottonseed powder. The mushroom fruiting bodies did not contain any residues of gossypol at concentrations of cottonseed powder 0.15–0.60% nitrogen contents of rice straw at the end of mycelial ramification. However, the cottonseed supplementation (at 0.30% N level itself) caused a doubling in the mushroom yield and its protein content, per unit weight straw substrate. The mushroom mycelium when grown on synthetic medium in liquid cultures was able to biodegrade gossypol. A pre-incubation period of 5 days before the addition of gossypol into the culture medium, an inoculum load 10 mg and an incubation period of 10 days at 25 °C caused the biodegradation of 100 g gossypol. Increased concentrations of gossypol required increased duration and increased inoculum levels to effect biodegradation. However, the effect was more pronounced with an increase in inoculum density. The fungal monoculture when grown in rice straw (powder) (5%) + glucose (1%) liquid culture medium, showed an increase in hexosamine content and laccase activity that produced an increased degradation of gossypol over an incubation period from 5 to 25 days. Enzymic extracts of the mycelial monoculture raised on the chopped rice straw substrate when incubated with 100 g of gossypol demonstrated its biodegradability; the increase in enzyme concentration showed enhanced gossypol degradation. This study adds to the world list of organic compounds that Pleurotus is able to biodegrade, and explains the cause of non-yellowing of the white oyster mushroom (P. florida) fruiting bodies, during culture on rice straw with supplementation of cottonseed powder for enhancing the mushroom yields.     

Comparative toxicity of Pfiesteria spp., prolonging toxicity of P. piscicida in culture and evaluation of toxin(s) stability

Toxicity of Pfiesteria piscicida (strain CAAE #2200) in the presence of fish (juvenile hybrid tilapia, Oreochromis sp., total length 3–6 cm) has been maintained in the laboratory for 19 months by serial transfer of toxic cells using a modified maintenance protocol. Toxicity was re-induced when toxin-producing P. piscicida cells were separated from fish and cultured on algal prey for 50 days and then re-introduced to new tanks containing fish. We confirmed toxicity in a strain of P. shumwayae (strain CAAE #101272). Toxicity to fish was demonstrated in culture filtrates (0.2 μm) derived from cultures of both Pfiesteria spp., however, it was markedly reduced in comparison to unfiltered water. Filtrates retained toxic activity when stored at −20 °C for up to 6 months. Toxicity to fish was retained when filtrates were held at room temperature for 48 h, at 70 °C for 30 min or at 88–92 °C for 2 h. P. piscicida killed all finfish species tested. Grass shrimp (Paleomonetes pugio; adult 2–3 cm), blue crab (Callinectes sapidus; juvenile 4–7 cm) and brine shrimp (Artemia sp.; 18–24 h post-hatch) were unaffected by concentrations of toxin(s) that killed juvenile tilapia in 4–24 h. Ichthyotoxic activity of filtrates from fish-killing cultures and stability of the toxic activity were similar among P. piscicida and P. shumwayae. These results confirm previously reported observations on toxicity of P. piscicidaand P. shumwayae to finfish. We have maintained toxicity in the laboratory for longer periods than have previously been routinely achieved, and we have demonstrated that the toxic activity is heat stable. In contrast to previous studies with other toxic P. piscicida strains, we did not observe toxic activity to blue crabs or other crustaceans.     

Cultivation of oyster mushrooms (<Emphasis Type="Italic">Pleurotus</Emphasis> spp.) on various lignocellulosic wastes

Cultivation of speciality mushrooms on lignocellulosic wastes represents one of the most economically and cost-effective organic recycling processes. Three species of Pleurotus, namely P. columbinus, P. sajor-caju and P. ostreatus were experimentally evaluated on untreated organic wastes including chopped office papers, cardboard, sawdust and plant fibres. Production studies were carried out in polyethylene bags of about 1 kg wet weight with 5% spawning rates of substrate fresh weight in a custom-made growth room especially designed for spawn run and cropping. The conversion percentage from dry substrate weight to fresh mushroom weight (biological efficiency) was determined. The highest biological efficiency was noted with P. columbinuson cardboard (134.5%) and paper (100.8%), whereas P. ostreatus produced maximum yield on cardboard (117.5%) followed by paper (112.4%). The overall yield of P. sajor-cajuwas comparatively low (range 47–78.4%). The average number of sporophore flushings ranged between 5 and 6 times. The findings that P. columbinus and P. ostreatus are superior to P. sajor-caju are consistent with previous reports elsewhere. Further evaluation of P. columbinus alone on different bagging systems containing partially pasteurized office papers as a growing substrate revealed that polyethylene bags resulted in 109.4% biological efficiency in contrast to pottery (86%), plastic trays (72%) or polyester net (56%). The above findings reveal an opportunity for commercial implication of oyster mushroom especially P. columbinus for utilization of different feasible and cheap recyclable residues.     

Effects of different levels of wheat bran, rice bran and maize powder supplementation with saw dust on the production of shiitake mushroom (Lentinus edodes (Berk.) Singer)

The cultivation of shiitake mushroom (Lentinus edodes) is increasing rapidly in Bangladesh due to its nutritional and medicinal importance with excellent flavor and longer shelf life. With the aim of increased production, we have cultivated L. edodes on saw dust (SD) supplemented with different levels (10%, 15%, 20%, 25%, 30%, 35% and 40%) of wheat bran (WB), rice bran (RB), maize powder (MP) and their combination (WB+RB+MP = 1:1:1) to investigate the growth, yield and quality of this mushroom. Most of the growth, yield and quality parameters varied significantly when mushrooms were cultivated with different levels of supplementation. The yield of mushroom was increased with the level of each supplementation upto a certain level, and then decreased. SD supplemented with 25% WB produced the highest number of fruiting bodies (34.8/500 g packet), highest biological yield (153.3/500 g packet), and biological efficiency (76.6%) of L. edodes. But the yield of the best quality mushroom was observed on SD with 40% WB supplementation; however, the qualities were not always supplementation dose dependent. In this study, we report that 25% WB supplementation with SD may be very effective for higher yield and 40% WB supplementation for better quality of L. edodes.     

Cultivation of Gracilaria verrucosa (Gracilariales,Rhodophyta) Strain G-16 for agar

The agarophyte, Gracilaria verrucosa Strain G-16, has been grown in sustained cultivation over a period of five years. During this period, a number of experiments were conducted to examine the productivity, agar yield and agar gel strength of this strain. Productivities range from 3–31 g dry wt m^–2 d^–1 and are generally highest in the summer when annual water temperatures and daily irradiances are highest. In the summer months agar yield from Strain G-16 appears to be lowest whereas the gel strength of the agars was highest (> 750 g cm^–2).