Rapid Mycelial Growth Research Articles

Assessment of Various Substrates for Shiitake Mushroom (Lentinula edodes) Cultivation in the Agro-Climatic Conditions of West Bengal

Aim: This study aimed to evaluate the impact of different substrates on various stages of shiitake mushroom (Lentinula edodes) cultivation to optimize growth and yield. Study Design: A Completely Randomized Design with three replications for each treatment was employed to ensure statistical reliability. Duncan's multiple range tests were utilized at a significance level of 5% to evaluate differences between treatments. Place and Duration of Study: The research was conducted at the Mushroom Research Laboratory, Department of Plant Pathology, Palli Siksha Bhavana (Institute of Agriculture), located in Sriniketan, Birbhum district, West Bengal, throughout the year 2023. Methodology: Different substrates including lentil husks, rice straw, wheat straw, sawdust, and mustard pods were evaluated for their impact on spawn run completion, mycelial bump formation, and sporocarp formation durations. Each substrate's performance was assessed in terms of sporocarp production and biological efficiency. Results: Across the three strains (LE-01, LE-02, and LE-03), lentil husks consistently exhibited the shortest spawn run duration, averaging 34.59 to 37.00 days. Following closely, wheat straw displayed favorable performance with durations ranging from 39.20 to 41.23 days. Sawdust also proved efficient, albeit slightly longer than lentil husks and wheat straw. Conversely, maize cobs and sugarcane bagasse exhibited longer spawn run durations, averaging from 57.80 to 64.90 days. For mycelial bump formation, lentil husks again showed the shortest duration, averaging 42.67 to 45.77 days across the strains. Wheat straw followed suit with durations ranging from 48.93 to 50.20 days, while sawdust demonstrated favorable times albeit slightly longer. Conversely, maize cobs and sugarcane bagasse exhibited longer mycelial bump formation times, ranging from 68.11 to 74.40 days. For sporocarp formation, lentil husks and sawdust emerged as top performers with the shortest durations, averaging 76.66 to 81.52 days. Conversely, maize cobs exhibited the longest sporocarp formation time, averaging 97.43 days. In terms of sporocarp yield, rice straw yielded the highest, averaging 27.67 sporocarps per bag, followed by wheat straw with 24.33 sporocarps per bag. Lentil husks, while efficient in other aspects, produced an average of 21.78 sporocarps per bag, trailing slightly behind rice straw and wheat straw. Regarding yield and biological efficiency, sawdust displayed superior performance, yielding an average of 384.57 grams per 750 grams of substrate with a biological efficiency of 51.18%. Lentil husks and rice straw exhibited comparable performance in yield and biological efficiency, with lentil husks yielding 367.81 grams and rice straw yielding 333.09 grams per 750 grams of substrate, both with biological efficiencies around 47-48%. Conversely, mustard pods exhibited the lowest yield and biological efficiency, averaging 269.02 grams per 750 grams of substrate with a biological efficiency of 35.91%. Conclusion: The study underscores the importance of substrate selection in optimizing various stages of L. edodes cultivation. Lentil husks emerged as a promising substrate choice for rapid mycelial growth, but its sporocarp production lagged behind other substrates. Sawdust proved to be the top-performing substrate in terms of yield and biological efficiency, indicating its efficacy in supporting robust mushroom growth.

First Report of Charcoal Rot Caused by Macrophomina phaseolina on Stevia rebaudiana in Arizona, USA.

Stevia (Stevia rebaudiana Bertoni) is an important medicinal crop grown worldwide. Leaves of stevia contain a non-caloric sweetener, stevioside, which is used as a substitute to artificial sweeteners. In August 2022, symptoms of chlorosis, wilting, and root rot were observed in about 30 % of stevia plants growing at the Agricultural Station at Yuma Agricultural Center, Yuma, AZ, USA (32.7125° N, 114.7067° W). Infected plants initially showed chlorosis and wilting, and the plants eventually died with foliage remaining intact to the plant. Cross sections of the crown tissue of affected stevia plants showed necrotic tissue and a dark brown discoloration in areas of the vascular and cortical tissues. Dark brown microsclerotia were observed on stem bases and necrotic roots of the infected plants. Five symptomatic plants were sampled to isolate the pathogen. Root and crown tissues (0.5 to 1 cm) were surface disinfested with 1% sodium hypochlorite for 2 min, rinsed three times with sterile water, and plated onto potato dextrose agar (PDA). All the five isolates displayed rapid mycelial growth on PDA at 28°C with a 12-h photoperiod. The mycelia were initially hyaline and turned from gray to black after 7 days. Masses of dark spherical to oblong microsclerotia were observed after 3 days on PDA, measuring an average of 75 µm width × 114 µm length (n=30). For molecular identification, genomic DNA was extracted from mycelia and microsclerotia of a representative isolate (Yuma) using the DNeasy Plant Pro kit (Qiagen, Hilden, Germany). The internal transcribed spacer (ITS), translation elongation factor-1α (TEF-1α), calmodulin (CAL), and β-tubulin (β-TUB) regions were amplified using the primer sets, ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), MpCalF/MpCalR (Santos et al. 2020), and T1/T22 (O'Donnell and Cigelink 1997), respectively. A BLAST search of sequences revealed 98.7 to 100% identity to Macrophomina phaseolina sequences (MK757624, KT261797, MK447823, MK447918). Both morphological and molecular characteristics confirmed the fungus as M. phaseolina (Holliday and Punithaligam 1970). Sequences were submitted in the GenBank under accession numbers OP599770 (ITS), OP690156 (TEF-1α), OP612814 (CAL), and OP690157 (β-TUB). Pathogenicity assay was performed on 9-week-old stevia plants (var. SW2267), grown in 4-inch planters in the greenhouse. The inoculum was made from a 14-day-old culture of M. phaseolina grown in conical flasks (250 ml) in potato dextrose broth at 28°C. Mycelial mats of the fungus were blended in 250 ml of sterile distilled water, filtered through four layers of cheesecloth, and then calibrated to 105 microsclerotia/ml using a hemocytometer. Twenty healthy plants were inoculated by soil drenching 50 ml of the inoculum per pot. Soil drenching using sterile distilled water was done on 5 non-inoculated control plants. Plants were maintained in the greenhouse at 28 ± 3°C with 12 h photoperiod. After 6 weeks, necrosis at the base of petioles and chlorosis of the leaves, followed by wilting were noticed on all 20 inoculated plants, whereas all the 5 control plants remained healthy. The fungus was reisolated and identified as M. phaseolina based on the morphology and sequences of ITS, TEF-1α, CAL and β-TUB regions. Although M. phaseolina has been reported earlier on stevia in NC, USA (Koehler and Shew 2018), this is a first report from AZ, USA. M. phaseolina is known to be favored by high soil temperatures (Zveibil et al. 2011), thus represents a potential threat to stevia production in AZ, USA in coming years.

Influence of Culture Media and Environmental Factors (Water Potential and Temperature) on Mycelial Growth of Phytopythium vexans (de Bary), the Causal Agent of Dieback Disease in Apple Trees

This study aimed at evaluating the effects of culture media and environmental factors (temperature and water potential (Ψw)) on the growth of the pathogenic fungus Phytopythium vexans (de Bary) associated with root rot and dieback disease in apple trees. Tomato agar, potato dextrose agar (PDA), and soybean agar were the most favourable for rapid mycelial growth, with optimum growth recorded for PDA medium. To determine the environmental conditions that promoted the development of this phytopathogen, the effects of temperature (5–30 °C), water potential (Ψw) (−15.54; −0.67 MPa) (0.89–0.995 aw), and their interaction were evaluated on the in vitro radial growth rates of the five isolates of P. vexans and on their latency phase (time period prior to growth). The results of this study showed that temperature, water potential, and their interaction had significant effects (p < 0.001) on the radial growth rates and latency phases of all tested P. vexans isolates. All isolates were able to grow throughout the temperature range (5 to 30 °C), with the maximum radial growth rate being observed at the highest temperatures, 25–30 °C. Growth was seen to be faster at −0.67 MPa (0.995 aw) at 25 °C and 30 °C. No growth was observed at Ψw < −5.44 MPa (0.96 aw), regardless of the temperature. It was found that the length of the latency phase depended significantly on both environmental factors. The longest latency phases (5 days on average) were recorded at a temperature of 5 °C and Ψw of −0.67 MPa (0.995 aw) and −2.69 MPa (0.98 aw), while the shortest latency phases were observed at a temperature of 30 °C and a Ψw of −0.67 MPa (0.995 aw), with an average of 0.2 days. The findings from this study could help to understand the impact of these environmental factors on the occurrence of diseases caused by P. vexans and more likely to design a reliable preventive control strategy based on the avoidance of conditions that play in favour of the phytopathogen.

Culture of Pleurotus ostreatus in pine shavings: isolation of strains and evaluation of their productivity

In order to obtain strains of the edible mushroom Pleurotus ostreatus that can be cultivated in pine wood shavings, 36 monosporic cultures were isolated and propagated in vitro, selecting 6 cultures of rapid mycelial growth in order to obtain interspecimen crosses and dikaryon-monokaryon mating. Eleven of the crosses obtained and their 6 parental strains were cultured in vitro in order to determine their growth in pine shavings and barley straw (control), selecting 6 strains (3 crosses, 3 parental strains) for sporome production in the two growth substrates. At in vitro level, significant differences were found between the mycelial areas of monokaryons and dikaryons in the treatments (time, substrate) evaluated. In terms of sporome productivity, the cultures in barley straw presented higher biological efficiency (22.6%-46.4%) and production rate (0.31%-0.77%) values than those in the pine shavings (3.3%-12% and 0.03%-0.11%, respectively). Sporome pileus diameters of 5 cm to 9.9 cm were the most frequent in both substrates. In general, the crosses surpassed their parents in both mycelial growth and sporome production, although further study is required in order to facilitate increased productivity of the strains in this potentially useful agroforestry residue.

First Report of Charcoal Rot Caused by Macrophomina phaseolina on Faba Bean in China

Faba bean (Vicia faba L.) is an important legume crop in China. Soil-borne charcoal rot, caused by Macrophomina phaseolina (Tassi) Goid, is a devastating disease of many crops, including legumes. In late March 2018, wilt symptoms similar to those of charcoal rot were observed in faba bean plants (‘Yundouzao 6’) in a field located in Jianshui County (23.38° N, 102.49° E), Yunnan Province, China. This disease caused serious yield loss of faba bean. Infected plants initially showed leaf chlorosis and wilting, and the plants eventually died with the leaves remaining attached. When affected plants were examined, the basal stem pith was rotted, and a brown discoloration was observed after splitting the roots and stems. Symptomatic plants were sampled to isolate the pathogen. Three fungal isolates were obtained (FMP8-1, FMP8-2, and FMP8-4) and displayed rapid mycelial growth on potato dextrose agar at 28°C with a 12-h light regime. The mycelia were initially hyaline and turned from gray to dark with age. Masses of dark oblong microsclerotia (46 to 96 × 61 to 129 µm) formed on the cultures 3 days after incubation. On the basis of these morphological characteristics, the three isolates were presumptively identified as M. phaseolina (Smith and Wyllie 1999). To confirm this identification, the three isolates were molecularly analyzed using primers specific for M. phaseolina: MpKF1/MpKR1, which yielded a 350-bp target fragment (Babu et al. 2007). Additionally, the internal transcribed spacer (ITS) regions of the three isolates yielded ∼600-bp PCR fragments using the universal primers ITS4/ITS5 (White et al. 1990), and the resulting sequences (accession nos. MH323404, MH323405, and MH323406) showed 99 to 100% identity with several known M. phaseolina strains using a BLAST algorithm-based analysis, including strains CIAT519 (KR012878) and CPC13084 (KF95698) isolated from Phaseolus vulgaris; CPC21464 (KF951750) and CPC21386 (KF951701) from Arachis hypogaea; CPC21514 (KF951775), CPC21517 (KF951778), and CPC21450 (KF951739) from Vigna unguiculatain; CBS231.33 (KF951631) from Saccharum officinarum; CJMR135 (MF480342) from Corchorus olitorius; and CPC13086 (KF951700) from Solanum melongena. Thus, this fungus has a broad host range. Both morphological and molecular characteristics confirmed that the three isolates were M. phaseolina. Pathogenicity tests were performed on 2-week-old faba bean seedlings using three inoculation methods (detached leaf, cut stem, and stem puncture) and conducted in three replications with five seedlings per replication (Keeling 1982). All inoculated leaves, stems, and plants showed necrotic symptoms 4 days after inoculation. No symptoms were observed on the control leaves, stems, or plants. M. phaseolina was reisolated from all inoculated leaves, stems, and plants, fulfilling Koch’s postulates. The three isolates were also pathogenic on other legume crops, including soybean (Glycine max), mungbean (Vigna radiata), pea (Pisum sativum), and grass pea (Lathyrus sativus). Although M. phaseolina has been reported on legume crops including soybean, mungbean, and adzuki bean in China, to our knowledge this is the first report of M. phaseolina causing charcoal rot on faba bean in China.

Production of liquid spawn of an edible mushroom, Sparassis latifolia by submerged fermentation and mycelial growth on pine wood sawdust

Rapid mycelial growth rate of liquid spawn could suppress contamination, which is significant to the mushroom industry. The aim of this study was to investigate the potential of producing liquid spawn of an edible mushroom, Sparassis latifolia SP-C by submerged fermentation under controlled conditions and to evaluate its ability to colonise pine sawdust substrate. Uniform design was adopted to quantitatively analyze the effects of fermentation medium on mycilial biomass production from S. latifolia, three independent variables including glucose, corn flour and glutinous rice flour were studied. The optimal medium for maximum biomass production was glucose 10%, corn flour 3%, glutinous rice flour 3%, fish peptone 0.2% and KH2PO4 0.3%, it was incubated at 23±2°C with agitation rate of 150rpm. The maximum S. latifolia SP-C biomass production of 11.96g/L was achieved after 15 days of fermentation. The liquid spawn led to quick mycelial growth (1.69mm/d) on sterile pine sawdust substrate compared to the traditionally sawdust spawn (1.23mm/d).

Lentinus squarrosulus (Mont.) mycelium enhanced antioxidant status in rat model.

AimLentinus squarrosulus is an edible wild mushroom commonly found in Asia. This species has several interesting features such as rapid mycelial growth, and hence has the potential to be used as food, functional food, and nutraceuticals. Our previous study shows that L. squarrosulus contains potent antioxidant compounds in vitro. This study aims to investigate the in vivo bioavailability of L. squarrosulus mycelium extract and its antioxidant effect on biomarkers of antioxidant defense and oxidative stress.MethodsWater extract of mycelial biomass of L. squarrosulus was analyzed for in vivo antioxidant effects, including cupric-reducing antioxidant capacity (CUPRAC), glutathione peroxidase (GPx), xanthine oxidase (XO), advanced oxidation protein products (AOPPs), and lipid hydroperoxides (LHPs) at 0 and 28 days. GPx and XO were also analyzed in liver homogenates. Normal Sprague Dawley rats were treated with 250 and 500 mg/kg of extract for 28 days.ResultsThe serum CUPRAC level increased after treatment with both concentrations, indicating that there was sufficient bioavailability of the extract which contributed to the total antioxidant capacity. GPx activity in both serum and liver was increased and this correlated with LHP level after treatment with 250 mg/kg of extract, but XO activity was significantly decreased after treatment with 500 mg/kg of the extract. Lack of difference between AOPP levels implied that there were no significant changes in oxidative damage of protein after treatment.ConclusionThis study clearly showed that L. squarrosulus mycelium antioxidant extract contains absorbable antioxidants that enter the circulating plasma and cause a significant acute increase in plasma antioxidant capacity. Thus, the water extract of L. squarrosulus mycelium, which can be obtained abundantly by liquid fermentation, may serve as an antioxidant ingredient in functional foods and nutraceuticals.

Potential of Endophytic Fungi of Barnyard Grass Weed for Biological Control of the Rice Sheath Blight Pathogen, Rhizoctonia solani Kühn

Endophytic fungi are group of microorganisms that colonize internal tissues of plants but do not cause any harm to their hosts. Although it has been reported that endophytic fungi from weeds have the potential as biological control agents against disease-causing pathogens of crops, the efficacy of those from barnyard grass weeds against sheath blight disease of rice caused by Rhizoctonia solani Kuhn has not yet been studied. This study determined the potential of endophytic fungi of barnyard grass weed (Echinochloa glabrescens Munro ex Hook. f.) as biological control agents of R. solani and identified the most effective of these endophytic fungi. Of 577 endophytic fungal isolates studied, rapid mycelial growth over R. solani was shown by sterile mycelia (SM EF-ds61-73, SM EF-ds375-97), EF-ds68-129, Geotrichum sp. EF-ds104-16 and Mucor sp. EFds158-2 in in vitro pre-screening tests. In dual culture tests, however, EF-ds68-129, Geotrichum sp. EFds104-16 and SM EF-ds375-97 exhibited mycelial growth increments of 17.0, 13.3 and 14.2 mm, respectively, resulting in 24% growth inhibition of R. solani. Closer observation revealed that only Geotrichum sp. EF-ds104-16 showed hyperparasitism through coiling and disruption of R. solani hyphae at 6 and 8 days after initial contact. Keywords—Endophytic fungi, barnyard grass, Echinochloa glabrescens, sheath blight, Rhizoctonia solani, weed, rice, biological control, hyperparasitism, dual culture

PENGARUH SUBSTITUSI LIMBAH CAIR INDUSTRI TAHU PADA MEDIA TANAM TERHADAP PERTUMBUHAN DAN PRODUKSI JAMUR TIRAM PUTIH (Pleurotus ostreatus) SEBAGAI SUMBER BELAJAR BIOLOGI

This study aims to get a dose of the best concentrations of liquid waste in order to produce rapid mycelial growth and production of white oyster mushrooms with optimal fresh weight. In this study using Analysis of Variance (ANOVA) One Direction with four treatments, each treatment be repeated six times. The treatments tested were (A) the concentration of liquid waste 25%/L water/ treatment, (B) the concentration of liquid waste 30%/L water/treatment (C) concentration of liquid waste 35%/L water/treatment and (D) control. The results showed that the concentration of industrial wastewater know 25%/L water / highest treatment effect on the growth and production of white oyster mushroom (Pleurotus ostreatus) with a mean weight of 129.33 g r fresh row A; B 105.5 g r ; C 76 . 17 g r ; and D 90.83 gr. The results could be used as a source of learning biology at the high school level with the subject matter of the class X Waste and Recycling Waste. In addition, this study also can be a reference for researchers and the general public. Kata Kunci : limbah cair, jamur, produksi, biologi .

Improvement in in vitro growth rates of Ganoderma species with industrial wood waste supplements

The bracket-like polypore fungi of the genus Ganoderma, as a pathogen of oil palm (Elaeis guineensis Jacq.), is a major concern primarily because it plays a significant role in the economy of many countries in South-East-Asia. Growth of several Ganoderma isolates was examined on various culture media to develop a medium for rapid growth. Growth analyses revealed that growth of the different isolates was different on various culture media which differed in their nutrient composition and their growth levels were influenced by industrial wood waste. High rate of growth was achieved on rubber/oil palm wood extract agar. Potato dextrose agar supplemented with wood powder and peptone also favoured mycelial growth. Malt extract agar was the poorest among the media investigated for obtaining rapid mycelial growth of Ganoderma isolates. G. boninense PER 71, G. tornatum POR 57 and G. subamboinense var. laevisporium (ATCC 52419) presented higher growth rate on the different culture media. The G. boninense PER 71 showed the fastest growth on the different media, while the minimum growth was achieved for G. tornatum POR 54. Rubber/oil palm wood extract agar media appears to increase growth rate of the different Ganoderma isolates. Increase growth rate of different Ganoderma isolates by industrial wood waste suggested that industrial wood waste is sufficient to improve mycelial growth of Ganoderma isolates. Supporting the assumption, industrial wood waste could be a useful renewable source for the early detection of Ganoderma disease that will improve efforts to prevent economic losses in oil palm. Our rapid and less expensive culture media may also have commercial potential and novel biotechnological applications.

Rapid enzyme production and mycelial growth in solid-state fermentation using the non-airflow box

Solid-state fermentation (SSF) has become an attractive alternative to submerged fermentation (SMF) for the production of enzymes, organic acids, and secondary metabolites, while there are many problems during the culture of SSF. We recently created a SSF system using a non-airflow box (NAB) in order to resolve the problems, which enabled the uniform culture in the whole substrate and high yield of many enzymes. In this paper, further characterization of SSF using the NAB was carried out to obtain other advantages. The NAB culture under the fixed environmental condition exhibited a rapid increase in enzyme production at earlier phase during the culture compared with conventional SSF. Total mycelial growth also exhibited the same trend as enzyme production. Thus, the increase in the rate of the enzyme production was thought to mainly be attributed to that of the growth. To support it, it was suggested that the NAB culture resulted in most optimal water activity for the growth just at the log phase. In addition, the NAB culture was able to achieve high reproducibility of enzyme production, derived from uniform condition of the substrate during the culture. The results indicate that the NAB culture has many benefits for SSF.

Morphological and molecular characterization of Colletotrichum coccodes isolated from pepper cultivated in Bulgaria and Macedonia

Colletotrichum coccodes has been recognized as one of the causal agents of pepper fruit anthracnose in Bulgaria. Recently, this species has been recorded in pepper fruits in Macedonia. In Bulgaria, the fungus has also been isolated from roots of premature senescent pepper plants but in Macedonia it has not been isolated yet. The purpose of the investigation was to make comparative morphological, cultural and molecular characterization of C. coccodes isolates obtained from pepper fruits and roots in Bulgaria and Macedonia. Additionally, a technique was applied to differentiate among the C. coccodes isolates obtained from roots and other microsclerotia-producing fungi. On the host tissue, C. coccodes developed acervular conidiomata with cup-shaped fruiting bodies accompanied with dark-pigmented, unbranched, thick-walled sterile hyphae called setae. A slimy mass of hyaline, straight, unicellular, fusiform conidia appeared on nutrient media. In a short time, numerous small dark globose setose microsclerotia emerged in the colony starting from its centre and distributing proportionally throughout agar plates. Two PCR primer sets were used to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions. Single products of ~450 bp and ~350 bp were amplified by the genus-specific (Cc1F1/Cc2R1) and the species-specific primers (Cc1NF1/Cc2NR1), respectively. Morphological, cultural and molecular characterization of the isolates from roots and fruits showed that root rot and fruit anthracnose of pepper were caused by one and the same causal agent determined as C. coccodes. The isolates from roots showed rapid mycelial growth, gave rise to numerous minute microsclerotia and produced conidia only on SSPA. SSPA supported significantly more mycelium growth and sporulation than all other media tested and could be recommended for production of large quantity of conidia. No pattern of genetic variation associated with the organ or geographic origin of the isolates was determined.

천연배지 열수추출물을 이용한 큰느타리버섯 균사배양 적합 배지 개발

본 연구는 버섯재배에 이용되는 배지재료를 이용하여 상업용 PDA 고체배지를 대체할 수 있는 원균 증식용 천연고체배지 제조 가능성에 대해 조사하였다. 천연고체배지는 큰느타리버섯 재배에 이용되는 재료를 열수 추출하여 제조하였다. 포플러톱밥, 밀기울, 미강이 버섯의 균사생육에 좋은 특성을 보였다. 천연 고체배지 제조를 위한 포플러톱밥, 밀기울, 미강의 적정 혼합 비율은 50 : 20 : 30(v/v/v, PWR523)와 50 : 30 : 20(v/v/v, PWR532) 수준이었다. 열수수출물 함유 천연 고체배지인 PWR523 및 PWR532에 대한 버섯 균사생육은 PDA 배지에 접종된 것보다 빠른 특성을 나타내었다. 또한 열수추출물 함유 천연 고체배지에 의해 만들어진 접종원을 이용한 경우 버섯의 배양 및 수확에 있어서 상업용 배지를 이용한 것과 큰 차이를 보이지 않았다. Hot-water extracted natural media were made from raw materials for mycelial culture of Pleurotus eryngii. Poplar sawdust, wheat bran and rice bran were used as substrates for hot water extraction. The mixed substrates of poplar sawdust, wheat bran, and rice bran with 50 : 20 : 30 (v/v/v, PWR523) and 50 : 30 : 20 (v/v/v, PWR532) were optimal for mycelial growth of P. eryngii, respectively. The hot-water extracted natural media from PWR523 and PWR532 showed a rapid mycelial growth and spawn running compared to PDA. There was no significant difference in mushroom yield when the mycelium grown on the hot-water extracted natural media was used as the inoculum source for producing fruit body.

Generating thermotolerant colonies by pairing Beauveria bassiana isolates

Low thermotolerance in entomopathogenic fungi is the main impediment to their industrialization. This research, for the first time, describes the generation of a thermotolerant colony by pairing and subculturing (cycling) two Beauveria bassiana isolates without sexual reproduction. A mixture of B.bassiana ERL1578 and ERL1576 was inoculated on quarter-strength Sabouraud dextrose agar with yeast extract (¼SDAY). The paired culture (ERL1578+1576) was cycled three times to increase the frequency of possible hyphal fusion at the first cycle (c.5/5×10(5) conidia), followed by a heat treatment as a selection pressure. Two non-paired isolates served as controls. Two morphologically different colonies (BbHet1 and BbHet2) were isolated from the pairing. BbHet1 colony had the highest conidial yield. BbHet2 had the most rapid mycelial growth and produced sponge-like mycelial masses (the others were flat), and its conidia were darker than the non-paired colonies under a microscope (400×). BbHet2 conidia had 60.7% germination after exposure to 45°C for 60min (the others had <15%) without significant loss of virulence against Western flower thrips, Frankliniella occidentalis; however, there was a slight decrease in conidial yield. The new phenotypes formed suggested that a genetic variation happened as a result of heterokaryosis and/or recombination, more than environmental adaptation, when mixing different conidia. This methodology seems to be very useful for enhancing thermotolerance in fungi.

Optimal Culture Conditions for Mycelial Growth of Lignosus rhinocerus

Lignosus rhinocerus is a macrofungus that belongs to Polyporaceae and is native to tropical regions. This highly priced mushroom has been used as folk medicine to treat diseases by indigenous people. As a preliminary study to develop a culture method for edible mushrooms, the cultural characteristics of L. rhinocerus were investigated in a range of culture media under different environmental conditions. Mycelial growth of this mushroom was compared on culture media composed of various carbon and nitrogen sources in addition to C/N ratios. The optimal conditions for mycelial growth were 30°C at pH 6 and 7. Rapid mycelial growth of L. rhinocerus was observed on glucose-peptone and yeast extract peptone dextrose media. Carbon and nitrogen sources promoting mycelial growth of L. rhinocerus were glucose and potassium nitrate, respectively. The optimum C/N ratio was approximately 10 : 1 using 2% glucose supplemented as a carbon source in the basal media.

Influence of culture media and environmental factors on mycelial growth and conidial production of Diplocarpon mali

To identify media and environmental conditions suitable for rapid mycelial growth and sporulation of Diplocarpon mali. Liquid shake cultures were used to evaluate effects of media and environmental conditions on mycelial growth and conidial production of D. mali. Carrot sucrose broth (CSB), potato and carrot dextrose broth (PCDB) and potato and carrot sucrose broth (PCSB) were most favourable for rapid mycelial growth. PCDB, PCSB, PCB (potato and carrot broth) and carrot dextrose broth (CDB) were favourable for conidial production. All carbon sources tested and peptone favoured for mycelial growth. Carbon and nitrogen sources tested did not significantly stimulate conidial production. The optimum temperature for mycelial growth and conidial production was 25 degrees C. No mycelial growth occurred at 5 or 30 degrees C, but D. mali survived at these temperatures. Active mycelial growth occurred at pH 5-7, and pH 5-8 was favourable for sporulation. PCDB and PCSB incubated at 25 degrees C for 14 day are recommended for mycelial growth and conidial production of D. mali. The information generated in this study will facilitate mycological and pathological research on D. mali and Marssonina leaf blotch of apple caused by D. mali.

Chemical properties and antioxidant activity of exopolysaccharides from mycelial culture of Cordyceps sinensis fungus Cs-HK1

Exopolysaccharide (EPS) was produced in a mycelial liquid culture of the Cordyceps sinensis fungus, Cs-HK1, at a rate proportional to mycelial growth. The crude EPS isolated from the culture medium by ethanol precipitation contained 65–70% sugar and about 25% protein, suggesting that the EPS was composed of polysaccharide–protein complexes. It exhibited a wide molecular weight (MW) range from about 5 kDa to more than 200 kDa. The intrinsic viscosity as an index of average MW of EPS, increased during the period of rapid mycelial growth to 11.0 dL/g on day 5, and remained relatively stable between day 5 to day 8 (stationary growth period). Both infrared spectrometry of EPS and gas chromatography of acetylated EPS suggested that the EPS had a β- d-glucan backbone. The EPS showed moderate antioxidant activities with a Trolox equivalent antioxidant capacity of 35–40 μmol Trolox/g and a ferric reducing ability of plasma of 50–52 μmol Fe(II)/g.

Pangola grass colonized with Scytalidium thermophilum for production of Agaricus bisporus

This work had the dual objective of selecting a substrate for rapid mycelial growth of Scytalidium thermophilum and then comparing the growth and production of a brown variety of Agaricus bisporus on substrate non-colonized and colonized with S. thermophilum. Mycelial growth of S. thermophilum at 45 °C was significantly greater on potato dextrose yeast extract agar (0.58 mm/h) as compared to malt extract glucose agar (0.24 mm/h) and yeast extract glucose agar (0.44 mm/h). On cereal grain, S. thermophilum grew significantly faster on rice (0.31 mm/h) compared to sorghum (0.22 mm/h) and millet (0.18 mm/h). It also grew faster on Pangola grass (0.49 mm/h) compared to corncobs (0.30 mm/h) and sawdust (0.18 mm/h). Colonization of Pangola grass with S. thermophilum was influenced by the addition of calcium salts in the form of gypsum, hydrated lime and ground limestone. For production of A. bisporus, biological efficiency (BE) on pasteurized Pangola grass pre-colonized by S. thermophilum for 4 days at 45 °C was more than twice (26.4%) that on grass non-colonized by S. thermophilum (11.0%). The addition of 2% hydrated lime to Pangola grass prior to colonization by S. thermophilum resulted in an additional doubling of BE of mushroom production (48.1%). These results show the possibility of developing a non-composted substrate method for producing A. bisporus without autoclaving the substrate.

The Characteristics of Cultural Conditions for the Mycelial Growth of Macrolepiota procera

Macrolepiota procera, one of edible mushrooms belongs to Agaricaceae of Basidiomycota, has a good taste and good medicinal value. As a preliminary study for the development of artificial cultivation method of edible mushroom, cultural characteristics of M. procera was investigated on various culture media under different environmental conditions. Mycelial growth was compared on culture media composed of various carbon and nitrogen sources, and C/N ratios. The optimal conditions for the mycelial growth were 30°C and pH 7. M. procera showed the rapid mycelial growth in the PDA media. The optimal carbon and nitrogen sources were maltose and glycine, respectively. The optimum C/N ratio was about 10 : 1 in case that 1% glucose was supplemented to the basal media as carbon source.

Effects of culture media and environmental factors on mycelial growth and pycnidial production of Potebniamyces pyri

Potebniamyces pyri (anamorph Phacidiopycnis piri) is the causal agent of Phacidiopycnis rot of apples and pears. The disease has recently been recognized in pears in the USA. Little information on the basic biology of the fungus is available. In this paper, we report the effects of culture media, temperature, water potential and pH on mycelial growth, and the effects of media and light on pycnidia production. Prune juice agar was the best for rapid mycelial growth. Pear juice agar, apple juice agar, potato dextrose agar, and oatmeal agar (OMA) also favoured mycelial growth. Czapek-Dox agar was not suitable for mycelial growth. The fungus was able to grow at temperatures from -3 to 25 degrees C. Optimal mycelial growth occurred between 15 and 20 degrees. The average radial growth rate on OMA was 3.9 mm d(-1) at 15 degrees and 4.4 mm d(-1) at 20 degrees. Mycelial growth was not observed after 10 d at 30 degrees, but growth resumed at 20 degrees. The fungus failed to resume growth at 20 degrees after being incubated for 10 d at 35 degrees. The fungus was able to grow at water potentials as low as -4 MPa but no growth took place at -7.3 MPa. Active mycelial growth was observed on OMA at pH between 3.2 and 6.1. Optimal growth was observed at pH around 4 and no growth was observed at pH 7.1. No pycnidia or very few formed on the nine media at 20 degrees in the dark. Fluorescent light significantly stimulated formation of pycnidia. OMA was the best medium for production of pycnidia and macroconidia. Pycnidia that formed on 8-wk-old OMA cultures incubated at 20 degrees under 12 h dark/12 h light produced abundant macroconidia and the technique is recommended for inoculum production of conidia for research.