Mycelial Growth Rate Research Articles

Transcription factor FfMYB15 regulates the expression of cellulase gene FfCEL6B during mycelial growth of Flammulina filiformis

BackgroundCellulose degradation can determine mycelial growth rate and affect yield during the growth of Flammulina filiformis. The degradation of cellulose requires the joint action of a variety of cellulases, and some cellulase-related genes have been detected in mushrooms. However, little is known about the transcriptional regulatory mechanisms of cellulose degradation.ResultsIn this study, FfMYB15 that may regulate the expression of cellulase gene FfCEL6B in F. filiformis was identified. RNA interference (RNAi) showed that FfCEL6B positively regulated mycelial growth. Gene expression analyses indicated that the expression patterns of FfCEL6B and FfMYB15 in mycelia cultured on the 0.9% cellulose medium for different times were similar with a correlation coefficient of 0.953. Subcellular localization and transcriptional activity analyses implied that FfMYB15 was located in the nucleus and was a transcriptional activator. Electrophoretic mobility shift assay (EMSA) and dual-luciferase assays demonstrated that FfMYB15 could bind and activate FfCEL6B promoter by recognizing MYB cis-acting element.ConclusionsThis study indicated that FfCEL6B played an active role in mycelial growth of F. filiformis and was regulated by FfMYB15.

Prevalence of H6Y mutation in β-tubulin causing thiophanate-methyl resistant in Monilinia fructicola from Fujian, China

Brown rot disease broke out in stone fruit orchards of Fujian, China in 2019, despite pre-harvest application of methyl benzimidazole carbamate (MBC). To determine the reason, a total of 44 Monilinia fructicola strains were collected from nectarine, plum and peach fruits in this study, among which 79.5% strains were resistant to thiophanate-methyl, indicated by discriminatory dose of 5 μg/mL. The resistance of these strains was confirmed by treating detached peach fruit with label rates of formulated thiophanate-methyl which only completely inhibit infection of the sensitive strains, but not the resistant strains. Further analysis of the mechanism of MBC resistance revealed that all resistant strains carry a H6Y mutation in β-tubulin protein Tub2, which was only reported previously in the M. fructicola strains from California, USA, and do not display obvious fitness penalties, as no significant defects in mycelial growth rate, sporulation, conidia germination, aggressiveness on detached peach fruit and temperature sensitivity was detected. In addition, we found that diethofencarb, the agent for managing MBC-resistance strains, was unable to inhibit growth of the H6Y strains. Taken together, our study, for the first time, identified a mutation form of H6Y in the β-tubulin protein of M. fructicola in China, rendering the strains wide resistance to thiophanate-methyl. This mechanism of M. fructicola gaining resistance to MBC fungicides needs to be fully considered, when designing management strategies to control brown rot disease in stone fruit orchards.

Synthesis of Novel Indole Schiff Base Compounds and Their Antifungal Activities.

A series of novel indole Schiff base derivatives (2a–2t) containing a 1,3,4-thiadiazole scaffold modified with a thioether group were synthesized, and their structures were confirmed using FT-IR, 1H NMR, 13C NMR, and HR-MS. In addition, the antifungal activity of synthesized indole derivatives was investigated against Fusarium graminearum (F. graminearum), Fusarium oxysporum (F. oxysporum), Fusarium moniliforme (F. moniliforme), Curvularia lunata (C. lunata), and Phytophthora parasitica var. nicotiana (P. p. var. nicotianae) using the mycelium growth rate method. Among the synthesized indole derivatives, compound 2j showed the highest inhibition rates of 100%, 95.7%, 89%, and 76.5% at a concentration of 500 μg/mL against F. graminearum, F. oxysporum, F. moniliforme, and P. p. var. nicotianae, respectively. Similarly, compounds 2j and 2q exhibited higher inhibition rates of 81.9% and 83.7% at a concentration of 500 μg/mL against C. lunata. In addition, compound 2j has been recognized as a potential compound for further investigation in the field of fungicides.

Synthesis, thermal property and antifungal evaluation of pyrazine esters

Synthesis, thermal properties, as well as antifungal assessment of pyrazine esters were handed in this work. The metal-free esterification of pyrazinyl methanol and acid chloride derivatives in a stoichiometric ratio yielded the following compounds: pyrazin-2-ylmethyl benzoate (3a), (5-methylpyrazin-2-yl) methyl benzoate (3b), 1-(pyrazin-2-yl) ethyl benzoate (3c), 2-(pyrazin-2-yl) ethyl benzoate (3d), pyrazin-2-ylmethyl pivalate (3e). The spectral results totally showed that the synthesis conditions used permitted the compounds to be produced with great yield. The characterization included 1H NMR, 13C NMR, IR, HRMS and organoleptic tests. Their thermal degradation process was examined by using the TG (thermogravimetry) and DSC (differential scanning calorimeter) methods. The pyrolysis products of the pyrazine esters in inert and oxidative atmospheres were analyzed by the Py-GC/MS (pyrolysis–gas chromatography/mass spectrometry) method. It revealed that 3a–3e were totally evaporated throughout the pyrolysis experiments at low temperatures. And they evaporated with high relative contents in pure nitrogen (86.12%–94.17%) and oxidative (75.01%–88.85%) atmospheres, with only a small amount decomposing into a series of substances. Additionally, the in vitro antifungal effects of compounds 3a–3e against R. solani, P. nicotianae, F. oxysporum, F. graminearum, and F. moniliforme were further investigated using the mycelial growth rate methodology. The findings shown that compound 3c has 94% and 80% inhibitor rates at 0.5 mg/ml against R. solani and P. nicotianae, respectively, with an EC50 value (half maximum effective concentration) of 0.0191 mg/ml and 0.1870 mg/ml, respectively. Similarly, that of compounds 3a and 3b exhibited 82% and 91% at 0.5 mg/ml against R. solani, with an EC50 value of 0.0209 mg/ml and 0.0218 mg/ml, respectively. The molecular docking of compound 3c with SDH (Succinate dehydrogenase) was preliminarily performed to reveal the binding modes in active pocket and analyze the interactions between the molecules and the protein.

Status of Fungicide Resistance and Physiological Characterization of Tebuconazole Resistance in Rhizocotonia solani in Sichuan Province, China.

The resistance prevalence of chemical fungicides has caused increasingly serious agro-ecological environmental problems. However, there are few previous reports about resistance to succinate dehydrogenase (SDHI) or sterol demethylation inhibitor (DMI) in Rhizoctonia solani, one of the main agro-diseases. In this study, the fungicide resistance of 122 R. solani isolates in Sichuan Province was monitored by the mycelial growth rate method. Results showed that all isolates were susceptible to hexaconazole and most isolates were susceptible to thifluzamide, except for the field isolate MSRS-2-7 due to a moderate resistance to thifluzamide (16.43-fold resistance ratio, RR), compared to the sensitivity baseline of thifluzamide (0.042 μg/mL EC50 values). On the contrary, many isolates showed moderate or high resistance to tebuconazole (10.59- to 60.78-fold RR), reaching EC50 values of 0.54~3.10 μg/mL, especially for a highly resistant isolate LZHJ-1-8 displaying moderate resistance to epoxiconazole (35.40-fold RR due to a 3.54 μg/mL EC50 value). The fitness determination found that the tebuconazole-resistant isolates showed higher fitness cost with these characteristics, including a lower growth rate, higher relative electric conductivity, an increased ability to tolerate tebuconazole, and high osmotic pressure. Four new mutations of cytochrome P450 sterol 14α-demethylase (CYP51), namely, S94A, N406S, H793R, and L750P, which is the target for DMI fungicides, was found in the tebuconazole-resistant isolates. Furthermore, the lowest binding energy with tebuconazole was also found in the LZHJ-1-8 isolate possessing all the mutations through analyses with Discovery Studio software. Therefore, these new mutation sites of CYP51 may be linked to the resistance against tebuconazole, and its application for controlling R. solani should be restricted in some areas.

Effect of the lignocellulose substrate type on mycelium growth and biocomposite formation by Ganoderma lucidum GA3P

The lignocellulose agricultural wastes, one of the major environmental pollutants, represent an extremely rich resource with high nutritional value, which can be used in the production of value-added products. In the current study the effect of different lignocellulose substrates on the growth rate of Ganoderma lucidum GA3P and the formation of mycelium-based bio-composites was determined. The macromorphology and specific mycelial growth rate of the colonies on different media containing various lignocellulosic substrate were studied. The obtained composites were characterized regarding their density of the mycelial growth, apparent density and size. G. lucidum GA3P demonstrated high μmax values ranging from 0.267 d-1 to 0.558 d-1 and low K values indicating that all used media were suitable for cultivation, but when wheat bran was used, the formed mycelium-based bio-composites possessed the best characteristics with highest apparent density recorded (0.39 ± 0.005).

First Report of Colletotrichum nymphaeae CausingWalnut Anthracnose in China.

Walnut(Juglans regiaL.) is a high quality woody nut and edible oil tree with a planting area of about 5,000,000 hectare in China. Walnut anthracnose is a serious disease, infecting approximately 50% of the fruits and causing a great yield losses (Wang et al. 2016). In 2019 to 2020, walnutfruits with anthracnose symptoms were collected from walnut orchards in province of Hubei, Sichuan procinve and Chongqing municipality, China. Symptoms on fruits were circular or subcircularor irregular shaped, with brown to black water soaked and sunken lesions. The black lesions enlarged and amalgamated into large necrotic areas. The older spots in the center became blackish with acervuli causing the complete mummification of the fruit, and orange conidial masses appeared under wet conditions. Necrotic tissues of the fruits were sterilized in 75% ethanol solution for 30 s, then sterilized in 4% sodium hypochlorite for 1min, and washed 3 times with sterile distilled water. The tissues were put on potato dextrose agar (PDA) and incubated at 25℃. Pure cultures were obtained by single-spore culturing method and the isolate HBBK4-4 was deposited into the China's Forestry Culture Collection Center (CFCC 57388). On PDA, the colonies were cottony, white to pale gray with aerial mycelium on the upper side and pink with black spots on the reverse. The mycelial growth rate was 9.6 mm/day at 25°C. Conidia were 1-celled, colorless, smooth-walled, straight, cylindrical to cylindrical-clavate with acute ends, 12.5 to 18.2 × 3.9 to 5.4 μm (mean 15.3 ± 3.7 × 4.9 ± 0.6 μm,n= 40). Most conidia germinated and developed one pleurogenous, 1-celled appressorium. Appressoria were single, medium brown, smooth-walled, ovate to ellipsoid, 5.4 to 7.8 × 5.4 to 7.8 μm (mean 6.7 ± 0.6 × 6.3 ± 0.5 μm,n= 30). These morphological characteristics were in concordance with published descriptions of Collectotrichum acutatum species complex. To further confirm the identity, internal transcribed spacer (ITS), beta-tubulin (TUB2), chitin synthase 1 (CHS-I) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified and sequenced (Damm et al. 2012). The ITS (OM189549) and TUB2 (OM273642) sequences of isolate HBBK4-4 showed 100% similarity, and GAPDH (OM249791) and CHS-1 (OM273641) sequences showed 98.7% and 99.6% similarity withC. nymphaeaeCBS100064 respectively. A maximum likelihood phylogenetic tree was generated based on combining all sequenced loci in MEGA5. 18 isolates including HBBK4-4 fell in theC. nymphaeaeclade with 96% bootstrap support. To verify Koch's postulates, six isolates were used for pathogenicity test, and 20 healthy fruits and 15 fully expanded leaves for each isolate were inoculated with 5-mm-diameter mycelial plugs. Controls consisted of detached premature fruits inoculated with a PDA plug without the fungus. Six days after inoculation, all fruits and leaves developed anthracnose symptoms similar to those observed in the field, while the controls remained healthy. The pathogenicity tests were repeated twice with the same results. The morphology of the reisolated fungi was consistent with the inoculated one, fulfilling Koch's postulates.The isolate HBBK4-4 was identified as C. nymphaeae, based on the description byDamm et al. (2012). The species C. nymphaeae has been previously reported to cause severe anthracnose on walnut in France (Da Lio et al., 2018), Brazil (Savian et al., 2019) and Italy (Luongo et al., 2022). To our knowledge, this is the first report ofC. nymphaeaeas a pathogen ofwalnutanthracnose in China. The result provided crucial information for epidemiologic studies and management of this disease.

Non-targeted metabonomics and transcriptomics revealed the mechanism of mulberry branch extracts promoting the growth of Sanghuangporus vaninii mycelium.

Sanghuangprous vaninii is a wood-inhabiting fungus, and its mycelium and fruiting body show excellent medicinal values. Mulberry is one of the major hosts of S. vaninii, however, the mechanism of mulberry affecting the growth of S. vaninii has not been reported. In the present study, a mulberry-inhabiting strain of S. vaninii was selected to explore the effects of mulberry branch extracts (MBE) on the growth of the strain. Results showed that MBE could significantly promote the growth of S. vaninii mycelium at the concentration of 0.2 g/l. After 16 days of liquid culture, the dry weight of mycelium in 0.2 g/l MBE medium was higher by three times compared with that in the control. The non-targeted metabonomic analysis of the culture medium at different culture times and concentrations was conducted to find the key components in MBE that promoted the growth of S. vaninii mycelium. Under the different concentrations of MBE culture for 10 and 16 days, 22 shared differential metabolites were identified. Next, in accordance with the peak value trend of these metabolites, HPLC–MS and liquid culture validation, four components derived from MBE (i.e., scopoletin, kynurenic acid, 3,5-dihydroxybenzoic acid and 2,4-dihydroxybenzoic acid) could significantly increase the growth rate of mycelium at the concentration of 2 mg/l. Transcriptomic and qRT-PCR analyzes showed that MBE could upregulate hydrolase-related genes, such as serine–glycine–asparaginate–histidine (SGNH) hydrolase, alpha-amylase, poly-beta-hydroxybutyrate (PHB) depolymerase, glycosyl hydrolase family 61, cerato-platanin protein and Fet3, which might enhance the nutrient absorption ability of S. vaninii. Importantly, MBE could significantly increase the content of harmine, androstenedione and vesamicol, which have been reported to possess various medicinal effects. Results suggested that MBE could be an excellent additive for liquid culture of S. vaninii mycelium, and these hydrolase-related genes also provided candidate genes for improving the nutrient absorption capacity of S. vaninii.

Synthesis and Antifungal Activity of New butenolide Containing Methoxyacrylate Scaffold.

In order to improve the antifungal activity of new butenolides containing oxime ether moiety, a series of new butenolide compounds containing methoxyacrylate scaffold were designed and synthesized, based on the previous reports. Their structures were characterized by 1H NMR, 13C NMR, HR-MS spectra, and X-ray diffraction analysis. The in vitro antifungal activities were evaluated by the mycelium growth rate method. The results showed that the inhibitory activities of these new compounds against Sclerotinia sclerotiorum were significantly improved, in comparison with that of the lead compound 3–8; the EC50 values of V-6 and VI-7 against S. sclerotiorum were 1.51 and 1.81 mg/L, nearly seven times that of 3–8 (EC50 10.62 mg/L). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observation indicated that compound VI-3 had a significant impact on the structure and function of the hyphal cell of S. sclerotiorum mycelium and the positive control trifloxystrobin. Molecular simulation docking results indicated that the introduction of methoxyacrylate scaffold is beneficial to improving the antifungal activity of these compounds against S. sclerotiorum, which can be used as the lead for further structure optimization.

Olive and green tea leaves extract in Pleurotus ostreatus var. florida culture media: Effect on mycelial linear growth rate, diameter and growth induction index

The exponential disposal of agro-industrial wastes onto the environment has endangered all forms of life. The implementation of these wastes in mushroom production is an eco-friendly and promising solution. The effect of olive and tea leaves extracts represented in culture media treatments: potato dextrose agar (PDA) 80% + tea extract (TE) 20% (T2), PDA 80% + olive extract (OE) 20% (T3), PDA 80% + OE 10% + TE 10% (T4), PDA 70% + OE 20% + TE 10% (T5) on mycelial growth diameter (MGD) and linear growth rate (MLGR) of Pleurotus ostreatus var. florida was compared to the one of PDA used as control (T1) at 22, 25 and 28°C inoculation temperatures. Optimum MGD was observed at 28°C in T1 (day 2), while it was significantly increased by 1.1–1.4 folds (days 4, 6, 8) in culture media containing plant extracts nevertheless the inoculation temperature. MLGR was improved by 102%–145% in olive/tea culture media compared to PDA nevertheless the inoculation temperature. Optimum growth induction index (GII) was observed in T3 (22.2%) at 22°C, T5 (21.9%) at 25°C and T4 (18.2%) at 28°C. These findings suggest the combination of olive and tea leaves extracts in the production of Pleurotus ostreatus.

Combinatorial Synthesis of Sulfonate Derivatives of α/β-Naphthol as Anti- Oomycete Agents.

Developing new, efficient, and environment-friendly small molecule fungicides is the key to effectively prevent and control plant pathogenic oomycetes. α/β-Naphthol is an important raw material for drug synthesis. Due to its special structure, α/β-naphthol and its analogs possess significant biological activity. The preparation and anti-oomycete activity of novel sulfonate derivatives based on α/β-naphthol against Phytophthora capsici have not been reported yet. Thirty-two sulfonate derivatives of α/β-naphthol (4a-p and 5a-p) were prepared. The structure of all title compounds was identified by 1H NMR, MS, and m.p. The anti-oomycete activity of 4a-p and 5a-p against P. capsici was determined using the mycelial growth rate method. With our ongoing research aimed at the discovery and development of fungicidal agents, 4a-p and 5a-p were designed and synthesized, and their anti-oomycete activity against P. capsici was evaluated in vitro. Two compounds 4a and 5a were found to have good anti-oomycete activity against P. capsici, and their corresponding EC50 values were 63.41 and 65.21 mg/L, respectively. It has been observed that the substituent R in these derivatives is aliphatic, which is very important for maintaining their anti-oomycete activity. The results lay a foundation for further design and development of sulfonate derivatives of α/β-naphthol as fungicidal agents. The structure- fungicidal activity relationship of α/β-naphthol derivatives is under investigation in our laboratory.

Characterization of ‘Cabernet Sauvignon’ pomace extracts and evaluation of antifungal potential of Alternaria sp. and Fusarium sp.

This study was conducted to capitalize on the waste produced by the vinification process which proved to be important sources of bioactive compounds with significant antifungal properties. ‘Cabernet Sauvignon’ grape pomace extracts were characterized in terms of total polyphenol content, antioxidant potential, but also evaluated in terms of antifungal effect against phytopathogenic fungi (Fusarium sp. and Alternaria sp.). The isolates used in this study were grown on three potato-dextrose-agar culture media, Czapek Dox and Malt-Agar. The highest amount of polyphenols was determined from the extract obtained by the microwave-assisted method (42.76 mg/g GAE), followed by the extract obtained by maceration (30.37 mg/g GAE). The lowest amount was obtained by the ultrasound-assisted method (15.06 mg/g GAE). However, the highest antioxidant activity was determined in the macerated extract, TEAC = 0.0523. The results of in vitro tests clearly indicated a high inhibitory percentage on the mycelium growth rate and, respectively, a significant decrease in spore germination power in Fusarium sp. (91.56%). However, for the pathogen Alternaria sp. further studies are needed to correctly validate the percentage of inhibition, as alcohol has been shown to have a negative effect on it.

A New Strain of Lecanicillium uredinophilum Isolated from Tibetan Plateau and Its Insecticidal Activity.

A new strain QHLA of Lecanicillium uredinophilum was isolated from a Chinese caterpillar fungus complex and its optimum growth temperature and fermentation conditions were studied. Its insecticidal activity was tested against larvae of seven different insect pests, including Henosepilachna vigintioctopunctata, Spodoptera exigua, Plutella xylostella, Spodoptera frugiperda, Sitobion avenae, Hyalopterus perikonus, and Aphis citricola. The optimum growth temperature was 21–24 °C. The highest spore production of the strain QHLA was 4.08 × 106 spore/mL on solid medium with a nitrogen source of NH4Cl. However, the highest mycelial growth rate of the strain QHLA was on solid medium with a nitrogen source from yeast extract (4.63 ± 0.03 mm/d). When the liquid medium contained peptone, yeast extract, and glucose, the water content of the mycelia was the lowest, while the spore production was the highest until day 12. When the liquid medium contained glucose, tussah pupa powder, KH2PO4, and MgSO4, the mycelia production was highest until day 8. The bioassay for insecticidal activity indicated that the LC50 values of QHLA were 6.32 × 103 spore/mL and 6.35 × 103 spore/mL against Sitobion avenae and Aphis citricola, respectively, while the LC90 values of the strain QHLA against Aphis citricola and Sitobion avenae were 2.11 × 107 spore/mL and 2.36 × 108 spore/mL, respectively. Our results demonstrated that the strain QHLA was a high virulence pathogenic fungus against insect pests, with the potential to be developed as a microbial pesticide.

Genetic, Phenotypic, and Pathogenic Variation Among Athelia rolfsii, the Causal Agent of Peanut Stem Rot in China.

Peanut stem rot caused by Athelia rolfsii is a serious soilborne disease worldwide and is becoming increasingly important in China. A total of 293 A. rolfsii isolates were collected from four representative peanut producing provinces in northern, central, and southern China. These isolates were assigned to 45 mycelial compatibility groups (MCGs) through pairing testing. The MCG diversity among isolates was greater in the southern sampled provinces compared with the northern provinces. A high level of genetic variability was found among the isolates from Guangdong Province in southern China. Variations were found in mycelial growth rate and sclerotial number, size, and dry weight of isolates sampled from places in different latitudes. Size and dry weight of sclerotia were positively correlated with latitude (P < 0.01), but the number of sclerotia was negatively correlated with latitude (P < 0.01). All tester isolates were pathogenic on peanut but varied in disease index. Inter-simple sequence repeat analysis and unweighted pair-group method with arithmetic average clustering resulted in three distinct clusters that were associated with the geographical location of the collection sites and sclerotial traits but were not associated with virulence of these isolates. These findings imply that genetic diversity, morphological traits, and virulence among A. rolfsii isolates varied in diverse geographical regions in China, and genetic diversity and sclerotial traits might be affected by latitude.

The peroxins BcPex8, BcPex10, and BcPex12 are required for the development and pathogenicity of Botrytis cinerea.

Peroxisomes have been proved playing roles in infection of several plant pathogens. Although the contribution of a portion of peroxins in pathogenicity was demonstrated, most of them are undocumented in fungi, especially, Botrytis cinerea. The homologs of Pex8, Pex10, and Pex12 in B. cinerea were functionally characterized in this work using gene disruption strategies. Compared with the wild-type strain (WT), the Δbcpex8, Δbcpex10, and Δbcpex12 mutants exhibited significant reduction in melanin production, fatty acid utilization, and decreased tolerance to high osmotic pressure and reactive oxygen species (ROS). The mycelial growth and conidiation of were significantly inhibited in Δbcpex8, Δbcpex10, and Δbcpex12 strains. The mycelial growth rates of Δbcpex8, Δbcpex10, and Δbcpex12 were reduced by 32, 35, and 34%, respectively, compared with WT and ectopic transformant (ET), and the conidiation was reduced by approximately 89, 27, and 88%, respectively. The conidial germination, germ tube elongation, and the formation of initiate infection structures (IFSs) were also reduced by the deletion of the genes. The pathogenicity was tested on the leaves of tobacco and strawberry, and fruits of tomato. On the leaves of tobacco and strawberry, the Δbcpex8, Δbcpex10, and Δbcpex12 mutants could not induce necrotic lesions, and the lesions on tomato fruits infected with the mutants were significantly reduced than those of the wide type. The results indicated that BcPEX8, BcPEX10, and BcPEX12 are indispensable for the development and pathogenicity of B. cinerea.

Influence of nutrient sources and environmental conditions on vegetative growth of fungus, Erythricium salmonicolor (Berk. &amp; Broome) Burdsall in cocoa

Erythricium salmonicolor is the cause of pink disease in cocoa in the humid tropics. The growth of 4 isolates (ES-WR006, ES-WR008, ES-ER009 and ES-ER010) of the fungus on different sources of nutrient (carbohydrate, amino acid and vitamin) and environmental conditions (temperature, pH and light) were evaluated in the laboratory. The pink disease isolates utilised the nutrients to different extent, with the best radial mycelial growth rate achieved with isolate ES-WR008 followed by ES-WR006. None of the isolates produced spores in the laboratory. The isolates utilised polysaccharides as best as disaccharides and monosaccharaides. Among 7 amino acids assayed, aspartic acid was the most promising for vegetative growth. The best radial mycelial growth on vitamins was attained on folic acid and riboflavin. The minimum, optimum and maximum temperatures for growing the fungus were 18 °C, 28 °C and 34 °C respectively. Survival of E. salmonicolor isolates within the temperature range of 18-34 °C is consistent with temperatures for growing cocoa in Ghana. The isolates also grew well within a pH range of 4-8, with the best growth at pH 6. Light quality and duration influenced the vegetative growth of the isolates. Pink disease isolates maintained in 24 hr darkness or 24 hr light performed better than in alternating 12 hr darkness and 12 hr light. There was a significant (p&lt;0.05) repressive effect of the alternating dark and light exposures on vegetative growth of the fungus. The implications of the results for efficient management of the cocoa disease are discussed.

ESTUDO DO CRESCIMENTO MICELIAL DE MACROPHOMINA SPP. NO BRASIL, SUA PATOGENICIDADE E SENSIBILIDADE A FUNGICIDA

ABSTRACT Macrophomina (Botryosphaeriaceae) is one of the main genera of soilborne phytopathogenic fungi, which causes root and seed rot in more than 800 host plants worldwide. Recent phylogenetic studies identified the species M. phaseolina and M. pseudophaseolina in Trianthema portulacastrum and Boerhavia diffusa in melon and watermelon production areas in northeastern Brazil. Therefore, the objective of this study was: i) to verify the effect of temperature and salinity on the mycelial growth of M. phaseolina, M. pseudophaseolina and M. euphorbiicola, ii) to assess their pathogenicity on melon and watermelon seedlings, and iii) to determine their sensitivity to the fungicide carbendazim. The optimal temperature for mycelial growth rate (MGR) for Macrophomina spp. ranged from 27.18 ºC (CMM4771 – M. pseudophaseolina) to 31.80 ºC (CMM4763 – M. phaseolina). For the effect of salinity on mycelial growth of Macrophomina isolates, the EC50 ranged from 103.76 (CMM4868 – M. euphorbiicola) to 315.25 mM (CMM4801 – M. pseudophaseolina). The pathogenicity test demonstrated that M. phaseolina, M. pseudophaseolina and M. euphorbiicola are pathogenic on melon with M. phaseolina exhibiting a higher level of virulence. Macrophomina euphorbiicola isolates did not cause disease in watermelon. The most sensitive isolates to the fungicide carbendazim were CMM4868, CMM4867 (M. euphorbiicola) and CMM1531 (M. phaseolina) with EC50 of 0.003, 0.012 and 0.012 mg.L-1 a.i., respectively. All Macrophomina spp. used in these experiments were pathogenic to the tested melon and watermelon cultivars with the exception of the M. euphorbiicola isolate that did not cause damage to watermelon.

First Report of Black Dot Caused by Colletotrichum coccodes on Potato in the Tibet Autonomous Region of China.

Potato black dot causing by Colletotrichum coccodes is a common disease in potato throughout the world, infecting underground stems, tubers, roots and foliage. Potato is becoming the third main food crop produced on ~16,000 ha annually in the Tibet Autonomous Region of China, situated on the world's highest plateau. However, the disease causing by C. coccodes has not been reported in this region. During the disease survey in the Bailang County of Tibet in August, 2020, some potato plants cv. "JiZhang 12" with chlorosis and wilting of foliage were observed. The incidence of affected plants was 20%. Necrotic lesions were also observed on the basal stems of the affected plants. Three affected plants were collected for pathogen isolation and three isolates were obtained for further investigation. The colonies on potato dextrose agar (PDA) were initially white, turning gradually black with age and producing abundant black sclerotia. Conidia were cylindrical, hyaline, aseptate, guttulate, with average size of 13.80 to 18.55×4.94 to 5.35 μm for the three isolates in which 30 conidia each were measured. Such characteristics are similar to C. coccodes (Lees and Hilton, 2003). Mycelial growth rate was 0.69 to 0.74 cm/day at 25 oC over the three isolates. The three isolates were confirmed to be C. coccodes by species-specific PCR using primer set of Cc1NF1/Cc2NR1 producing 350 bp amplicons in the internal transcribed spacer (ITS) region according to Cullen et al. (2002). The Cc1NF1/Cc2NR1 sequences were identical for three isolates, therefore one sequence from isolate BL_JZ_J1 was submitted to the GenBank with accession number OM368349. Additional genes were also sequencing including the actin (ACT), chitin synthase l (CHS1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and another larger ITS region were also amplified from genomic DNA using primers sets ACT512F/ACT783R, CHS-79F/CHS-354R (Carbone and Kohn 1999), GDF1/GDR1 (Templeton et al. 1992) and ITS1/ITS4 (Glass and Donaldson 1995), respectively. Sequences obtained for those four regions were 216 bp, 218bp, 235bp and 576 bp, respectively. Each region in the three isolates were also identical, therefore one sequence for each region was submitted to the GenBank with accession number of OM417059, OM417060, OM417061, and OM349570, respectively, which had 100% similarity with C. coccodes of MN336525 (ACT), KU821274 (CHS1), KU821397 (GAPDH) and KU821175 (ITS), respectively. Phylogenetic analyses based on the concatenated sequences of those four loci showed that the BL_JZ_J1 was close to C. coccodes, a reference isolate of CPOS1 with the accession numbers of GQ856787 (ACT), GQ856723 (CHS1), GQ856744 (GAPDH) and GQ485588 (ITS) (Yang et al. 2009). Pathogenicity was confirmed by inoculating a conidia suspension (100 µl of 105 conidia/ml) on three stems of 6-week-old potato plant cv. 'Favorita' with an artificial wound generated by sterile toothpick for each isolate. An equal volume of sterile water was injected on the wound of three stems as a noninoculated control. Brownish necrotic lesions were observed on all inoculated stems 3 days post-inoculation under natural conditions, whereas control stems remained symptomless. Reisolation of the fungus from all inoculated symptomatic plants confirmed Koch's Postulates. To our knowledge, this is the first report of C. coccodes in potato in Tibet Autonomous Region of China. The finding of black dot in this region has important management implications for the growers since the pathogen can survive for long periods in the field both on potato debris and in soil.

Excessive Oxalic Acid Secreted by Sparassis latifolia Inhibits the Growth of Mycelia during Its Saprophytic Process.

Sparassis latifolia is an edible and medicinal mushroom in Asia commercially cultivated on substrates containing pine sawdust. Its slow mycelial growth rate greatly increases the cultivation cycle. In this study, we mainly studied the role of oxalic acid (OA) secreted by S. latifolia in its saprophytic process. Our results show that crystals observed on the mycelial surface contained calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) according to X-ray diffraction (XRD). Vegetative mycelia secreted large amounts of OA during extended culture periods. However, high concentrations of OA decreased the mycelial growth rate significantly. Moreover, the degradation of lignocellulose was significantly inhibited under high concentrations of OA. These changes could be attributed to the significantly decreased activities of lignocellulose-degrading enzymes. In conclusion, by establishing a link between OA secretion by the mycelium and the slow growth rate of its saprophytic process, this work provides fundamental information for shortening the cultivation cycle of S. latifolia.

Combinatorial Synthesis of Novel 1-sulfonyloxy/acyloxyeugenol Derivatives as Fungicidal Agents.

Developing the high-efficiency and low-risk small-molecule greenfungicide is the key to effective control of the plant pathogenic oomycetes. Essential oils play a very important role in novel fungicide discovery for their unique sources and potential target sites. Eugenol, a kind of plant essential oil, was mainly isolated from the unopened and dried flower buds of Syzygium aromaticum of the Myrtaceae family. Due to its unique structural skeleton, eugenol and its derivatives have exhibited a wide range of biological activities. However, a study on the synthesis of novel 1-sulfonyloxy/acyloxyeugenol derivatives as fungicidal agents against Phytophthora capsici has not yet been reported. Twenty-six novel 1-sulfonyloxy/acyloxyeugenol derivatives (3a-p and 5a-j) were prepared and their structures were well characterized by 1H NMR, HRMS, and m.p. Their fungicidal activity was evaluated against P. capsici by using the mycelial growth rate method. To find novel natural-product-based fungicidal agents to control the plant pathogenic oomycetes, we herein designed and synthesized two series of novel 1-sulfonyloxy/acyloxyeugenol derivatives (3a-p and 5a-j) as fungicidal agents against P. capsici Leonian, in vitro. Results of fungicidal activity revealed that, among all compounds, especially compounds 3a, 3f, and 3n displayed the most potent anti-oomycete activity against P. capsici with EC50 values of 79.05, 75.05, and 70.80, respectively. The results revealed that the anti-oomycete activity of eugenol with the sulfonyloxy group was higher than that with the acyloxy group. It is suggested that the fungicidal activity of eugenol can be improved by introducing the sulfonyloxy group. This will pave the way for further design, structural modification, and development of eugenol derivatives as fungicidal agents.