PDA Plates Research Articles

First Report of Lasiodiplodia theobromae and L. pseudotheobromae Causing Canker Disease of Sacha Inchi in Hainan, China.

Sacha inchi (Plukenetia volubilis L.) belongs to the family Euphorbiaceae. It is a perennial wooden oilseed crop, and also exhibits a good source of polyunsaturated fatty acids, protein and other bioactive compounds, such as tocopherols, carotenes and phytosterols (Chirinos et al. 2013). During 2017-2018 survey, canker disease showing greyish-brown sunken lesions was observed on the branches of sacha inchi in Danzhou campus, Hainan University, China. The disease incidence is less than 5%. However, it can lead to leaf yellowing, wilt, and eventually the whole plant death. In Nov. 2017, twelve branches showing the typical canker symptoms were collected and covered with parafilm at both ends of all samples to prevent desiccation and placed in black plastic bags keeping at 4°C until isolations were made. Samples were rinsed with tap water and dried with paper towels. Fragments, 5mm in length and cut from the junction of diseased and healthy parts of branches, were surface-sterilized with 2% sodium hypochlorite solution for 2 min, rinsed with sterilized distilled water for 5 times, dried by sterilized filter paper, plated on PDA medium amended with 100 μg/mL streptomycin (PDA-str) and incubated in the dark for 4 days at 28°C. Pure cultures of fungal isolates were obtained by transferring mycelial fragments from colony margins onto fresh PDA plates and incubated as described before. The colonies of cultures were initially white, and eventually turned black after 4 days on PDA medium (Fig S1A). The morphology characterization of conidia produced by the isolates was initially hyaline and aseptate (Fig S1B), and a single median septum formed in the mature conidia (Fig S1B). The average size of 50 conidia was 16.39±1.46ⅹ 8.52±0.92μm for J6, and 15.64±1.73ⅹ 8.94±0.86μm for J3. Three genes were used for phylogenetic analysis (Alves et al. 2006). ITS regions and the partial of TUB (β-tubulin gene) were amplified using the primer pairs ITS1 and ITS4 (White et al. 1990), Bt2a and Bt2b (Glass and Donaldson 1995), respectively, and EF1-688F/EF1-1251R for J3 and EF1-728F/EF1-986R for J6 were used to amplify TEF (translation elongation factor 1-alpha) (Alves et al. 2008). The sequences of ITS, TUB and TEF from J3 and J6 were deposited in Gene-Bank (Table S1). The blast searches in Gene-Bank with ITS, TUB and TEF amplified from isolates J3, respectively, revealed 100, 99, and 100% identities with L. pseudotheobromae, and isolate J6 showed 100, 100 and 99% of identity with L. theobromae. The phylogenetic analysis of the combined ITS, TUB and TEF sequences of J3, J6 and 28 reference strains retrieved from Gene-Bank was performed using the program MEGA 6.0 evaluated by 1000 bootstrap replications, and the result was consistent with the conclusion above (Fig S2). With the phylogenic studies supported by morphological characters, J3 was identified as L. pseudotheobromae and J6 was L. theobromae. For the pathogenicity test, J3 and J6 were used to inoculate 4-week-old healthy sacha inchi potted seedlings. One wound about 5 mm in depth per seedling stem was made using a sterile blade. A 5-mm-diameter mycelium plug of each isolate taken from the edge of 4-day-old culture growing on PDA was placed to the freshly wound of each plant stem and the inoculated area was wrapped with Parafilm. Sterile PDA plugs were placed onto the wounds of control seedlings. Nine healthy seedlings were inoculated with each isolate or PDA plugs in a completely randomized design. After inoculation, plants were placed in a greenhouse at room temperature (26 to 30°C, 80% RH) and were irrigated when needed. The experiment was conducted twice. Five days later, black or dark-brown canker lesions formed on the stems of inoculated plants, and expended upward and downward from the inoculation points. Pycnidia produced on the necrotic regions and were used to to observe the morphology of conidia (Fig S3). The fungus L. pseudotheobromae or L. theobromae can be re-isolated from the inoculated plants, but not from the control ones. L. pseudotheobromae was recorded to be collected from dead leaves of P. volubilis in Yunnan Province, China, but did not prove this fungus to be pathogenic (Tennakoon et al. 2016). This is the first report that L. theobromae and L. pseudotheobromae causing stem canker in sacha inchi in Hainan, China. The results pave the way for the development of management strategies for canker disease in sacha inchi.

First Report on Colletotrichum siamense Causing Anthracnose of Castor Bean in Zhanjiang, China.

Castor bean (Ricinus communis L.) is an important oil crop. Anthracnose lesions were observed on leaves of castor bean at the stage of budding and fruiting in field (21˚17'51''N, 110˚18'16''E), Zhanjiang, Guangdong Province, China in August 2019. The incidencerate was approximately 40% (n=600 investigated plants). Early symptoms were yellow spots appearing from the edge or the tip of the leaves. Later, the spots gradually expanded and became dark brown, which coalesced into larger irregular or circular lesions (Supplementary Figure 1). Seven diseased leaves were collected from seven plants. Margins of the diseased tissue were cut into 2 mm × 2 mm pieces. The surfaces were disinfested with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Thereafter, the samples were rinsed three times in sterile water, placed on PDA, and incubated at 28 °C. Pure cultures were obtained by transferring hyphal tips to new PDA plates. A single-spore isolate (RLC-1) was used for further study. The colony of isolate RLC-1 on PDA was white to gray in color with cottony mycelia in 6 days at 28 °C. Conidia were one-celled, hyaline, cylindrical, clavate, obtuse at both ends and measured 14.2 to 18.5 µm × 3.8 to 5.5 µm (n =50). Appressoria were oval to irregular in shape, dark brown, and ranged from 7.3 to 10.5 µm × 5.7 to 6.5 µm (n = 20). Morphological characteristics of isolate RLC-1 were consistent with the description of Colletotrichum siamense (Prihastuti et al. 2009;Sharma et al. 2013). DNA of the isolate RLC-1 was extracted for PCR sequencing using primers for the rDNA ITS (ITS1/ITS4), GAPDH (GDF1/GDR1), and ACT (ACT-512F/ACT-783R) (Weir et al. 2012). Analysis of the ITS (accession no. MN880199), GAPDH (MN884048), and ACT (MN891766) sequences revealed a 99%-100% identity with the corresponding ITS (JX010250), GAPDH (KX578786), ACT (JX009541) sequences of C. siamense in GenBank. A phylogenetic tree was generated on the basis of the concatenated data from sequences of ITS, GAPDH, and ACT that clustered the isolate RLC-1 with C. siamense with the type strain ICMP 19118 (Supplementary Figure 2). Morphological characteristic and phylogenetic analysis identified the isolate RLC-1 associated with anthracnose of castor bean as C. siamense. Pathogenicity test was performed in a greenhouse at 24 °C to 30 °C with 80% relative humidity. Twenty healthy plants of Zi Bi No. 5 castor bean (2 month old) were grown in pots with one plant in each pot. Inoculation was conducted on leaves with mycelial plugs of RLC-1 or agar plugs (as control). Three plugs were considered for each leaflet. Ten plants were used in each treatment (five for wounded inoculation and five for unwound inoculation). Anthracnose lesions as earlier were observed on the leaves after 2 weeks, while the control plants remained healthy. The pathogen re-isolated from all inoculated leaves was identical to the isolate RLC-1 by morphology and ITS analysis but not from control plants. C. siamense causes anthracnose on various plant hosts, including mango in Colombia (Pardo-De la Hoz et al. 2016) and Rosa chinensis in China (Feng et al. 2019) but not including castor bean. To the best of our knowledge, this study is the first to report C. siamense causing anthracnose on castor bean. Thus, this work provides a basis for focusing on the management of the disease in future.

Study on the antifungal effect and mycolytic activity of the biocontrol agent Chaetomium subaffine LB-1

The antifungal effect and mucolytic activity of a newly screened biocontrol strain Chaetomium subaffine LB-1 were researched in this study. The results found that LB-1 has good antifungal effects on the test plant pathogenic fungi Botrytis cinerea Pers. ex Fr., Fusarium oxysporum f. sp. cucumerinum Owen and Alternaria solani (Ellis & Martin) Sorauer in dual culture assay, with the inhibition rates of 61.39, 50.34 and 51.22%, respectively. Flocculated and dissolved hyphae of the phytopathogenic fungi were observed at the interaction zone on a dual-cultured PDA plate, but the hyphae of LB-1 were normal. The LB-1 cell-free filtrate has significant inhibitory effects on the three tested fungi in the poison plate assay; dissolved colonies, vesiculated and flocculated hyphae of the test pathogenic fungi were also found on the PDA plates supplemented with the LB-1 cell-free filtrate. Clear halo zones around the LB-1 colonies were found on the protease test plate, pectinase test plate and cellulose test plate, indicating that LB-1 could produce mucolytic enzymes of protease, pectinase and cellulase. However, the activities of chitinase and β-l,3-glucase were not detected on their test plates from LB-1. An obvious oil-displaced circle was formed in the oil spreading test, indicating that a surface-active substance might be contained in the LB-1 cell-free filtrate. These results proved that the biocontrol agent of C. subaffine LB-1 could exert its antifungal effects via living competition and mycolysis, and the latter may be obtained by production of mycolytic enzymes and surface-active substances. .

Critical Evaluation of Two Commercial Biocontrol Agents for Their Efficacy against B. cinerea under In Vitro and In Vivo Conditions in Relation to Different Abiotic Factors

The study evaluated the dose–response relationship of two commercial microbial biocontrol agents, Bacillus subtilis and Gliocladium catenulatum, against Botrytis cinerea both in vitro and in vivo. Inoculum doses, formulation, temperature and foliar leaf part all affected the control achieved by the two BCAs. In vitro competition assays on modified PDA plates tested a range of BCA doses (log10 3–10 CFUs or spores/droplet) at 4, 10 and 20 °C on the development of B. cinerea colonies. The dose–response relationship was influenced by both the BCA formulation and temperature. In vivo studies on lettuce plants in semi-commercial greenhouses examined the BCA dose (log10 5–9 CFUs or spores/mL) for controlling B. cinerea with a high inoculum (log10 6 spores/mL). Leaf disc assays showed that the dose–response relationship was influenced by the leaf parts sampled. These results suggest that the dose–response relationship between a BCA and specific pathogen will be significantly influenced by environmental conditions, formulation and plant phyllosplane tissue.

First Report of Epicoccum tobaicum Associated with Leaf Spot on Flowering Cherry in South Korea.

Flowering cherry (FC, Prunus x yedoensis Matsumura; Somei-yoshino cherry) is an ornamental tree, planted across South Korea and producing stunning flowers in spring. The seasonal blooms are annually celebrated during cherry blossom festivals in many locations across the country. The leaf spot disease is among the most common and important diseases affecting FC trees every year, resulting in premature defoliation and reduced flowering of cherry blossoms in the following year. In May 2018, brown spots (2 to 5 mm), circular to irregular and with dark borders were observed on FC leaves in Hadong, Gyeongsangnamdo, South Korea (35°07'48.9"N, 127°46'53.8"E), with a disease incidence of 55%. Single lesions often coalesced and were sometimes perforated, leaving shot holes. Sampled leaves were surface sterilized with 1% NaOCl for 1 min and 70% ethanol for 30 s, and then rinsed twice with sterile distilled water. About 2-mm-long infected leaf pieces from the margins of lesions were put onto water agar (WA, 1.5% agar) plates and incubated at 25oC for 72 h. Mycelia grown from symptomatic tissue were transferred to PDA plates, and five similar fungal isolates were obtained from hyphal tips. They produced a strong reddish-orange diffusible pigment on PDA after 5 d and exudates after 8 d. Conidia were globular to pear-shaped, dark, verrucose, multicellular, and 14.8 to 23.5 μm in diameter (av. = 18.7 μm, n = 30) for isolate JCK-CSHF10. These morphological characteristics were consistent with the Epicoccum genus. Three loci, ITS, tub2, and rpb2, from three isolates JCK-CSHF8, JCK-CSHF9, and JCK-CSHF10 were amplified using the primer pairs ITS1F/LR5 (Gardes and Bruns 1993; Vilgalys and Hester 1990), Btub2Fd/Btub4Rd (Woudenberg et al. 2009), and RPB2-5F2/RPB2-7cR (Liu et al. 1999; Sung et al. 2007), respectively. The ITS, tub2, and rpb2 sequences of the three isolates were deposited in Genbank (MW368668-MW368670, MW392083-MW392085, and MW392086-MW392088, respectively), showing 99.6 to 100% identity to E. layuense (E33), a later synonym for E. tobaicum (Hou et al. 2020). The phylogenetic tree using concatenated sequences of the three loci placed the three isolates in a cluster of E. tobaicum (CBS 232.59, CGMCC 3.18362, and CBS 384.36; Hou et al. 2020). Taken together, the three isolates were identified as E. tobaicum. The pathogenicity of JCK-CSHF10 was tested on 15 healthy leaves on three FC trees (cv. Somei-yoshino, 1.2 m in height) kept in a greenhouse. Five-mm-diameter plugs from 7-d-old fungal cultures grown on PDA or mycelia-free PDA plugs as controls were placed on the abaxial side of a leaf at three points, previously wounded by a sterile needle (Zlatković et al. 2016). Inoculation sites were covered with moist cotton plugs. Trees were then covered with a clear plastic bag and maintained in high humidity at 25oC in darkness for 24 h, followed by a 12-h photoperiod. Brown spots appeared on inoculated leaves after 7 d, identical to those observed in the field, while control leaves remained symptomless. This experiment was repeated three times. A fungus with the same morphology as JCK-CSHF10 was recovered from lesions, thus confirming Koch's postulates. E. layuense (syn. E. tobaicum) has been reported as a leaf spot-causing agent on Perilla sp. (Chen et al. 2017) and Camellia sinensis (Chen et al. 2020). To date, there is no report on the occurrence of E. tobaicum from leaf spots on FC. To our knowledge, this is the first report of E. tobaicum causing leaf spot on FC in South Korea.

Pre-Symptomatic Disease Detection in the Vine, Chrysanthemum, and Rose Leaves with a Low-Cost Infrared Sensor

Thermography is a technique based on infrared imaging, which is used nowadays to detect plants under stress caused by biotic and abiotic factors. In many cases, temperature changes have already been correlated with pathogen attacks. In this sense, thermography offers the ability of early disease detection in plant pathology. In this work, a low-cost AMG8833 Grind-Eye infrared camera combined with a 1080P RGB web camera was used to develop an integrated infrared and RGB imaging system, to record temperature changes on vine, chrysanthemum, and rose plant leaf surfaces. Vine and chrysanthemum leaves were infected with Phomopsis viticola and Septoria ssp. respectively, respectively, whereas rose plants leaves were infected with Colletotrichum spp. as well as with Podosphaera pannosa. Measurements were performed using the integrated imaging system on infected and uninfected leaves, as well as on PDA plates with active and non-active mycelium. According to the results, vine leaf tissue infected with P. viticola and rose plants leaf tissue infected with P. pannosa had a pre-symptomatic (four days after infection) decrease in temperature up to 1.6 and 1.1 °C, respectively, compared with uninfected tissue. In contrast chrysanthemum leaf tissue infected with Septoria ssp. and rose plant leaf tissue infected with Colletotrichum spp. had a pre-symptomatic (four days after infection) increased temperature up to 1.1 °C and 1.0 °C, respectively, compared with uninfected tissue. In vitro measurements showed that the active fungi mycelium had approximately 1.1 to 2.1 °C lower temperature than the non-active mycelium. The results above show that the integrated infrared and RGB imaging system developed in this work can be used to detect early disease infection before visible symptoms appeared, facilitating the decision-making process.

Analytical study on Fungal Cellulase Produced by Penicillium Expansum grown on Malus Domestica (Apple Fruits)

The rise in world industrialization and the cost of importing enzyme by local industries have led to arise in the search for novel and native enzyme producing microorganisms. Cellulase is an enzyme that catalyzes the breaking down of carbon chains in cellulose and hemicellulose, this research therefore aimed at studying fungal cellulase produced by Penicillium expansum grown on malus domestica (apple fruits). Fresh apple fruit was allowed to deteriorate under laboratory condition until there was visible mould growth. The mould with desired features of the organism of interest was subcultured by direct plating on PDA plates to which 10 % streptomycin has been added to prevent bacterial contaminants. The plates were incubated at 28±2 0C for 7 days until a visible mass of blue mycelia appear. The isolate was further subcultured onto freshly prepared media until pure culture was obtained. Characterization and identification of isolate were done using macroscopy and microscopy techniques. The isolate was re-inoculated into healthy apple fruits and the fruits were incubated at temperature of 28±2 oC for 8 days. Cellulolytic activity was examined every day throughout the incubation period. Crude enzyme was extracted each day using standard methods. Carboxyl methyl cellulose was used as standard for the crude cellulase activity assay after extraction from the infected apple fruits using Dinitrosalicylic acid (DNSA). Culture parameters like pH and temperature were also optimized to determine their effect on cellulolytic activity of the fungus. Cellulase activity was defined as the amount of glucose produced in μmol/mg/min under the assay condition. The highest cellulase activity of 86.84±0.52 μmol/mg/min was observed on day 6 of incubation at 28±2 oC and at pH 7. In conclusion, it is evident from this research that P. expansum isolated could be used as potential novel organism for industrial production of cellulase under optimized fermentation conditions.

Volatile organic compounds produced by Pseudomonas fluorescens ZX as potential biological fumigants against gray mold on postharvest grapes

Volatile organic compounds (VOCs) produced by antagonistic microorganisms have the potential for controlling postharvest diseases on fruit and vegetables. In this study, the biocontrol efficacy of VOCs emitted by Pseudomonas fluorescens ZX was determined in vitro and in vivo against gray mold, caused by Botrytis cinerea, on grapes. In vitro, application of VOCs produced by bacterial suspension of P. fluorescens ZX at initial inoculum concentration of 1 × 109 colony-forming units (CFU)/mL inhibited B. cinerea mycelial growth from 52 to 23 mm and spore germination from 88.56 to 39.67%. In vivo tests indicated that, for grapes, disease incidence and disease index were markedly reduced by VOCs from P. fluorescens ZX incubated on NA plates, in NB, and on healthy grapes. Pre-fumigation with VOCs could inhibit development of gray mold on grapes. Furthermore, P. fluorescens ZX-producing VOCs exhibited favorable effects on postharvest natural decay of grapes. In vitro testing of selected pure chemicals demonstrated that dimethyl trisulfide (20 μL/L), dimethyl disulfide (40 μL/L), geranyl formate (640 μL/L), acetic acid (160 μL/L), butyric acid (320 μL/L), 2-methylbutyric acid (160 μL/L), isobutyric acid (160 μL/L) and isovaleric acid (320 μL/L) could completely inhibit mycelial growth of B. cinerea on PDA plates. These eight substances also exerted a strong inhibitory effect on gray mold on grapes. Additionally, SEM analysis showed that VOCs could affect the morphological and structural characteristics of B. cinerea both in vitro and in vivo. Collectively, VOCs produced by P. fluorescens ZX are promising for biocontrol of gray mold on grapes through fumigant action.

A Novel Hexose Transporter ChHxt6 Is Required for Hexose Uptake and Virulence in Colletotrichum higginsianum.

Colletotrichum higginsianum is an important hemibiotrophic plant pathogen that causes crucifer anthracnose worldwide. To date, some hexose transporters have been identified in fungi. However, the functions of hexose transporters in virulence are not clear in hemibiotrophic phytopathogens. In this study, we identified and characterized a new hexose transporter gene named ChHxt6 from a T-DNA insertion pathogenicity-deficient mutant G256 in C. higginsianum. Expression profiling analysis revealed that six ChHxt genes, ChHxt1 to ChHxt6, exhibited specific expression patterns in different infection phases of C. higginsianum. The ChHxt1 to ChHxt6 were separately deleted using the principle of homologous recombination. ChHxt1 to ChHxt6 deletion mutants grew normally on PDA plates, but only the virulence of ChHxt4 and ChHxt6 deletion mutants was reduced. ChHxt4 was required for fungal infection in both biotrophic and necrotrophic stages, while ChHxt6 was important for formation of necrotrophic hyphae during infection. In addition, ChHxts were functional in uptake of different hexoses, but only ChHxt6-expressing cells could grow on all five hexoses, indicating that the ChHxt6 was a central hexose transporter and crucial for hexose uptake. Site-directed mutation of T169S and P221L positions revealed that these two positions were necessary for hexose transport, whereas only the mutation Thr169 caused reduced virulence and defect in formation of necrotrophic hyphae. Taken together, ChHxt6 might regulate fungal virulence by modulating the utilization of hexose.

First Report of Lasiodiplodia theobromae Causing Branch Dieback on Castor Bean in Zhanjiang, China.

Castor bean (Ricinus communis L.) is an oil crop of significant economic importance in the industry and medicine. In August 2019, a branch dieback disease was observed on castor bean in a field in Zhanjiang (21.17°N, 110.18°E), China. The incidencerate was 35% (n=600 investigated plants). Symptoms were discoloration of leaves, branch dieback, and discoloration of internal stem tissues. The disease had spread to the whole branches and causing the plant to die. Seven diseased branches were collected from seven plants. Margins between healthy and diseased tissues were cut into 2 mm × 2 mm pieces. The surfaces were disinfested with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Then, the samples were rinsed thrice in sterile water, placed on PDA, and incubated at 28 °C. Pure cultures were obtained by transferring the hyphal tips to new PDA plates. Eighteen isolates were obtained (the isolate rate of 75%), which were the same fungus on the basis of morphological characteristics and molecular analysis of the internal transcribed spacer (ITS). A single representative isolate (RiB-1) was used for further study. The colony of RiB-1 was 5 cm in diameter on the 5th day on the PDA culture. The colony was greenish gray with an irregularly distributed and fluffy aerial mycelium, which turned black after 10 days. The mature conidia were 21.3-26.5 µm × 12.2-15.7 µm in size (n=100) and had two ovoid, dark brown cells with longitudinal striations. The morphological characteristics of the colonies were consistent with the description of Lasiodiplodia sp. (Alves et al. 2008). Three regions of the ITS, translation elongation factor (EF1-α), and β-tubulin genes were amplified and sequenced with the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Alves et al. 2008), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The resulting sequences were deposited in the GenBank under accession numbers MN759432 (ITS), MN719125 (EF1-α), and MN719128 (β-tubulin). BLASTn analysis demonstrated that these sequences were 100% identical to the corresponding ITS (MK530052), EF1-α (MK423878), and β-tubulin (MN172230) sequences of L. theobromae. Based on the morphological and molecular data, RiB-1 was determined as L. theobromae. A pathogenicity test was performed in a greenhouse with 80% relative humidity at 25 °C to 30 °C. Ten healthy plants of Zi Bi No. 5 castor bean (1-month-old) were grown in pots with one plant in each pot. Five pots were wound-inoculated with 5-mm-diameter mycelial plugs obtained from 7-day cultures. Five additional pots treated with PDA plugs served as the controls. Inoculated stems were moisturized with sterile cotton for five days. The test was conducted three times. Disease symptoms, similar to those in the field, were observed on the inoculated plants two weeks after inoculation, and L. theobromae was 100% reisolated from the inoculated plants. The control plants remained symptomless, and reisolations were unsuccessful. These results consistent with Koch's postulates. L. theobromae (Lima et al. 1997) and L. hormozganensis (Fábio et al. 2018) had been reported to cause stem rot on castor bean in Brazil, but whether L. theobromae caused the branch dieback on castor bean in China has not been reported yet. Thus, this study is the first report of L. theobromae causing the branch dieback on castor bean in Zhanjiang, China. This study provides an important reference for the control of the disease.

Micromelanconis kaihuiae gen. et sp. nov., a new diaporthalean fungus from Chinese chestnut branches in southern China.

Melanconis-like fungi are distributed in several families of Diaporthales, mainly Juglanconidaceae, Melanconidaceae, Melanconiellaceae and Pseudomelanconidaceae. A new Melanconis-like genus of Pseudomelanconidaceae was discovered on branches of Chinese chestnut (Castanea mollissima) in southern China, which was confirmed by both morphology and phylogenetic analysis of combined ITS, LSU, tef1a and rpb2 sequences. The new genus Micromelanconis is characterized by two types of conidia from natural substrate and manual media of PDA, respectively. Conidia from Chinese chestnut branches are pale brown, ellipsoid, multiguttulate, aseptate with hyaline sheath. While conidia from PDA plates are pale brown, long dumbbell-shaped, narrow at the middle and wide at both ends, multiguttulate, aseptate, and also with hyaline sheath. All Pseudomelanconidaceae species were only reported on tree branches in China until now. More interesting taxa may be discovered if detailed surveys on tree-inhabiting fungi are carried out in East Asia in the future.

Isolation, characterization, culturing, and formulation of a new Beauveria bassiana fungus against Diaphorina citri

The Asian citrus psyllid, Diaphorina citri, is a worldwide destructive citrus pest. It aids in the transmission of a group of phloem-limited bacteria (Candidatus Liberibacter spp.) that leads to Huanglongbing (HLB) disease. In this study, a new Chinese native fungal strain (F-HY006) was isolated from the cadavers of D. citri collected from Ganzhou, Jiangxi in China (114°94′ E, 25°87′ N). The isolate was virulent towards adult female D. citri with corrected mortality of 76% and LT50 of 4.09 d after application of a suspension containing 107 conidia/ml (400 µl/citrus seedling). Morphologic and phylogenetic analyses identified this isolate as Beauveria bassiana. Based on its virulence against D. citri and growth characteristics (2.89 ± 0.28 × 105 conidia/cm2) on the PDA plate, biphasic (liquid–solid) cultivation for conidia production of B. bassiana F-HY006 was optimized using several nutritional and environmental factors. Soybean meal + cottonseed powder (1:1) and continuous fluorescent light resulted in the highest conidial production (5.7 × 109/g dry substrate). Moreover, the oil-based conidial formulation was optimized using sesame oil as an effective carrier, which resulted in a significant enhancement of the conidial shelf life, tolerance to thermal stress, and virulence against D. citri compared to unformulated conidia. Therefore, we have prepared a potential mycopesticide for the biological control of D. citri based on the optimized biphasic culture growth and formulation of an isolated virulent fungus, B. bassiana F-HY006.

First report of Diaporthe eres causing branch canker on Cinnamomum camphora (camphor tree) in Jiangxi Province, China.

In September 2019, approximately 75 to 90% of camphor trees (Cinnamomum camphora) were observed with cankers and branch dieback symptoms in Anyi (N28°32'54'', E115°37'52'') and Xinyu (N27°37'38'', E114°50'25'') county (Jiangxi Province, China). The symptoms included dark brown to dark, oval-shaped canker lesions, sunken and cracked longitudinally, cracked and evenly swelling, or reddish brown (Figure 1 A-D). Samples were collected from symptomatic branches and were cut into small pieces (ca. 0.5 cm × 0.5 cm × 0.5 cm). Sections were surface sterilized as described by Zhang et al. (2020), then placed on potato dextrose agar amended with 0.01% penicillin and 0.015% streptomycin sulfate and incubated in the laboratory at 25℃ with darkness. After 3 to 5 days, mycelium growing out from tissues were transferred onto PDA medium. In total, 68 fungal isolates including 22 isolates of Diaporthe sp. were obtained from cankers and then were classified into five categories based on morphological characteristics and sequencing of the ITS for morphological representative strains. Pathogenicity tests were conducted in the greenhouse (Figure 1 E-M) and field (Figure 1 N-Q). Branches were surface sterilized and inoculated as described by Prencipe et al. (2017). In the greenhouse, a total of 13 representative isolates (including 6 isolates of Diaporthe sp., 2 isolates of Neofusicoccum sp., 2 isolates of Botryosphaeria sp. and 3 isolates of Colletotrichum sp.) were selected and evaluated using 2-year-old seedlings of camphor tree in pots with 5 replicates per isolate, in which 3 isolates of Collectotrichum sp. had no pathogenicity. Then, two isolates of Diaporthe sp. (Z4 and Z7) were selected for field experiment. In field tests, the same method was used as in the greenhouse. The inoculated and control branches were collected 40 days after inoculation and the fungi were isolated and placed on PDA plates to recover the inoculated fungi and complete Koch's postulates. Both isolates of Diaporthe sp. produced canker symptoms on the branches. Isolate Z4 caused discoloration also on the branch without wounding. Both isolates produced pycnidia scattered in PDA plates supplemented with stems of alfalfa, were dark brown to black, globose to subglobose (Figure 1 T). Alpha conidia were cylindrical, 5.72-9.98 µm (mean 7.64 µm) × 2.15-3.13 µm (mean 2.69 µm) (n = 30) (Figure 1 S, red arrow), while beta conidia were biguttulate, one-celled, hyaline, non-septate, and 16.21-25.52 µm (mean 21.60 µm) × 0.76~1.65 µm (mean 1.14 µm) (n = 30, green arrow) (Figure 1 S). Five isolates (Z4, S-Z4, P-Z4, Z7 and S-Z7) including those used for pathogenicity test were selected for multi-locus phylogenetic analyses of ITS (White et al., 1990), TEF1-α and TUB2 (Glass et al. 1995) gene sequences, which the accession number was MW036358- MW036362 for ITS, MW052267- MW052271 for TEF1- α, MW052276-MW052280 for TUB2. Based on the phylogenetic tree analysis using IQ-TREE 2, all five isolates were identified as D. eres (Figure 2). D. eres has been reported to cause canker on many different woody plants, such as almond (Holland et al. 2020), peach (Prencipe et al. 2017), hazelnut (Wiman et al. 2019), and so on. However, this is the first report worldwide of D. eres causing disease on Cinnamomum camphora in China.

Study of Coffee Thread Blight Disease (Corticium koleroga (Cke) Hoehnel) in Ethiopia

Thread blight caused by Corticium koleroga is a devastating disease that causes severe damage to Coffea arabica in Ethiopia. However, it is one of the least researched diseases in Ethiopia. Therefore, the current work was designed with the objectives to (1) assess the intensity of the disease, (2) identify major factors associated with the disease intensity, (3) identify and characterize C. koleroga isolates and (4) determine the pathogenicity of C. koleroga isolates in southwest Ethiopia. For this purpose, field surveys were conducted on 180 farmers’ fields in 12 districts of southwest Ethiopia during 2017 cropping season. In addition, diseased samples were collected from 11 districts of southwestern Ethiopia during the study. The 11 C. koleroga isolates were characterized using macroscopic and microscopic features. The isolates were cultured on potato dextrose agar and incubated at 25 oC for ten days. The treatments were arranged in a completely randomized design with three replications. The average thread blight incidence varied from 0% to 46% while mean disease severity ranged from 0% to 44%. The thread blight disease incidence and severity were higher at areas representing highland and midland altitudes compared with lowland altitudes. Thread blight severity was positively and strongly associated with rainfall (r = 0.75) and relative humidity (r = 0.85), but the disease severity was negatively and strongly associated with mean temperature (r = -0.79) and shade level (r = -0.50), while it was positively and intermediately correlated to altitude (r = 0.44) and coffee production systems (r = 0.36). The colony color of C. koleroga isolates were from white to floral white, with circular to irregular form and filiform to entire in margin on PDA plates. Growth rate of the isolates varied between 6 and 9 mm/day in diameter. Average basidiospore size ranged from 10 to 13.75 × 3.75 to 5 μ. All isolates were pathogenic to C. arabica with different lesion sizes. The present study revealed the importance of coffee thread blight, identified factors associated with the disease intensity and morphological characteristics of C. koleroga existing in southwestern Ethiopia. Future research should be directed towards surveying more agro-ecologies to have a complete picture on the importance of thread blight disease across the country and molecular characterization of the pathogen to recommend disease management strategies.

Diversity of Endophytic Fungi Isolated from <i>Gentiana rhodantha</i> and Screening for Biocontrol Stains

In order to clarify the composition of endophytic fungi in Gentiana rhodantha in colonization tissues with four Liupanshui sampling sites (Fenghuang Zhongshan, Nankai Shuicheng, Yangmei Shuicheng and Xinchang Liuzhi) in Liupanshui, strains were isolated with health leaf, stem and root and identification based on morphological characteristics and ITS sequence analysis. As well as screening for biocontrol stains by the dural culture method on PDA plates with Sclerotinia sclerotionrum . The results showed that G. rhodantha concealed rich fungal diversity, the colonization rate and isolation rate of endophytic fungi reached 49.38% and 61.25% respectively. 294 strains of endophytic fungi were obtained from 480 explant tissue segments of G. rhodantha and belonging to 2 phylum, 5 classes, 14 orders and 32 genera; of which Fusarium and Colletotrichum were comparatively advantageous taxa in endophytic fungi of G. rhodantha , and the species contained occupied 9.86% and 8.84% of the total endophytes respectively．According to the calculation results, Xinchang LZ in four sites and stem in three tissues showed optimum degrees of diversity, evenness and dominance respectively. 23 strains of endophytic fungi with inhibitory activities against S. sclerotionrum by the dural culture method; Amomg them, the inhibition rate of Botryosphaeria sp. R022, R021, Phomopsis sp. S002, Colletotrichum sp. S028 and Fusarium sp. L031 were higher than 70% and showed the best inhibitory potential.

Changes in Cytochrome b Gene Expression in Cochliobolus sativus Induced by Triadimefon, a Triazole Fungicide

Introduction: Spot blotch, caused by Cochliobolus sativus, is most effectively managed using fungicide applications, including triadimefon (TDM) a triazole compound. C. sativus posses a great concern as it might develop resistance against fungicides like TDM due to its high genetic variability, short life cycle, and abundant inoculum production. Therefore, to better understand the mechanisms of TDM resistance initiated by C. sativus, changes in cytochrome b (cytb) gene in virulent and avirulent pathotypes were evaluated at early time points of TDM treatments. Materials and Methods: C. sativus sensitivity to TDM was determined by measuring the radial growth of each pathotype on PDA plates. Additionally, RNA was isolated from mycelia of each pathotype at 24, 48, 72 and 96 hours post fungicide treatments and used for cDNA synthesis. Cytb was verified using quantitative reverse transcriptase PCR (qRT-PCR). Results: Data showed that the maximum mycelial growth inhibition by 50% (EC50) for both pathotypes was recorded 48h at 0.25 µg/ml TDM treatment. The qRT-PCR revealed that cytb expression increased in both virulent and avirulent pathotypes at 24h post TDM treatments in comparison with non-treated controls. The most outstanding differences in cytb expression were7.69 and 2.88-fold in the virulent and avirulent pathotypes, respectively, 48h of 0.25 µg/ml TDM treatment. Conclusions: According to findings, it is possible to propose that cytb gene might play a role in signaling events during C. sativus exposure to commercial triazole fungicide.

Fungal Tolerance: An Alternative for the Selection of Fungi with Potential for the Biological Recovery of Precious Metals

The behavior of various filamentous fungi in the presence of metals such as Cu, Zn, Ni, Fe, Mn, and V has been widely reported. However, there is little information regarding metals such as Au, Ag and Pt that are not in the form of nanoparticles. The growth of eight filamentous fungi was evaluated at increasing doses of Au, Ag and Pt. The fungi were reactivated in Petri dishes with potato dextrose agar. Subsequently, individual mycelial disks from each strain were inoculated in PDA plates with the following doses of AuCl3, Ag2SO4 and PtCl4: 0, 50, 150 and 300 mg L−1, respectively. The plates were then incubated for 20 days—a period in which the diameter of the colony was measured every 24 h. Au showed the highest toxicity for the tested fungi. All silver doses decreased the growth of most of the fungi, while platinum did not cause any inhibitory effect on the growth of the eight tested fungi. With a simple test, it was possible to observe the effect of precious metals (PMs) on the growth of filamentous fungi and consider their possible biotechnological applications in the recovery of PMs from primary or secondary sources.

Isolation Of Anticancer Drug Taxol Producing Endophytic Fungus Alternaria Alternata By Using HPLC And LC- MS

Abstract Introduction/Objective In the exploration of an alternative source, optimum and numerous solvents with different percentage were exploited to extract Taxol from plant palm branche. The fungal endophytes producing secondary metabolites that are effective against the human infections have fascinated many researchers across the world. Among these, the exploration for novel cancer therapeutic agents is of countless reputation due to upsurge in the number of cancer deaths, high worth of the drugs and the side effects concomitant with cancer treatments. Accordingly, in the current study, an endophytic fungal strain was isolated from its host plant palm branche and identified as Alternaria Alternata based on its morphological features and internal transcribed spacer sequence analysis. Methods Firstly, palm branche were collected for the isolation of endophytic fungi. The samples were placed on PDA plates and after 72 h, the mycelium produced were isolated and sub-cultured. Secondly, further analysis for the presence of Taxol by using chromatographic and spectrometric techniques. Illustratively, ethanol or acetone and 50% and 20% of ethanol. Then, the residues were dissolved in 5 ml of methanol and filtered. By using the internal standard, the resulting solution was subjected to the HPLC to determine the extraction efficiencies of tested solvents. Finally, filtrate was also subjected to the LC-MS – ESI using water/acetonitrile (10/90) as mobile phase to identify the authenticity of Taxol Results HPLC analysis revealed that Taxol could be quantified by UV detection using standard curve. Furthermore, the standard curve covering the concentration was linear (r2= 0.9992) and CV% ranged from 0.52 to 15.36. The peaks of fraction 5 obtained by HPLC analysis were quite similar to that of standard taxol confirming the presence of taxol. A parent ion peak of m/z 851.85 was observed in the LC–ESI–MS spectrum which was similar to paclitaxel with reported m/z of 854 [M+H] + ion. A. Alternata produced about 113.193 mg/L taxol as quantified through HPLC Conclusion we are recording for the first time the production of taxol by an endophytic fungus Alternaria Alternata. In a final word, present study results advocate that the endophytic fungus A.Alternata aids as a potential source for the production of taxol isolated from Palm branche plant

Biochemical and molecular investigation of non-rhizobial endophytic bacteria as potential biofertilisers.

This study was performed to isolate non-rhizobial endophytic bacteria from the root nodules of Glycine max (soybean), Vigna radiata (mung bean) and Vigna unguiculata (cowpea). The bacteria were characterized for plant growth promoting properties such as indole acetic acid production, phosphate and zinc solubilisation, nitrogen fixation and hydrogen cyanide production. Phylogenetic identification was performed using the Neighbour-Joining method on16S rRNA gene sequences. The impact of salt tolerant isolates on some properties of wheat cv. Chamran was evaluated by a completely randomised factorial design. Nine isolates having some characteristics related to plant growth promotion were identified as Staphylococcus hominis 7E, Streptomyces sp. 11E, Bacillus sp. 13E, Acinetobacter sp. 19E, from mung bean, Bacillus endophyticus 1E from cowpea, Staphylococcus hominis 9E, Bacillus endophyticus 14E, Brevundimonas sp. 16E and Kocuria sp. 26E from soybean nodules. Isolates 7E and 19E caused maximum growth inhibition of Fusarium on PDA plate. All isolates were able to grow at salinity levels of mixtures containing up to 400mM of NaCl, CaCl2 and MgCl2, but their growth was inhibited by increasing salinity level. Only the growth of isolate 14E increased at three levels of salinity compared with control. Some isolates, i.e. 7E, 14E, 19E and 26E had higher colony diameter at 45°C after 48h of incubation compared to the growth at 30 and 40°C. Inoculation of soil with isolate 1E and isolate 26E caused to ameliorate salinity stress in wheat and increased the weight of 1000-grains as compared with non-inoculated treatments.

Baseline Sensitivity and Toxic Action of the Sterol Demethylation Inhibitor Flusilazole Against Botrytis cinerea.

In the present study, a total of 95 Botrytis cinerea single-spore strains collected from different hosts in Shaanxi Province of China were characterized for their sensitivity to the sterol demethylation inhibitor fungicide flusilazole. The effective concentration for 50% inhibition of mycelial growth (EC50) of flusilazole ranged from 0.021 to 0.372 µg/ml, with an average value of 0.093 µg/ml. Cross-resistance between flusilazole and commonly used fungicides was not detected, and no flusilazole-resistant mutants were induced. Both on detached strawberry leaves and in greenhouse experiments, flusilazole was more effective than the commonly used fungicide carbendazim at reducing gray mold. After culture on PDA plates or detached strawberry leaves, no difference in sclerotia production or pathogenicity was detected between two strains, WG12 (most sensitive to flusilazole) and MX18 (least sensitive to flusilazole). After treatment with flusilazole, however, the two strains lost the ability to produce sclerotia, and oxalic acid and ergosterol contents in mycelium decreased. Interestingly, the inhibition rate of ergosterol content in MX18 was significantly lower than that in WG12. Expression of Cyp51, BcatrD, and Bcmfs1 genes all increased after treatment with flusilazole, especially the Cyp51 and BcatrD genes. However, the expression of Cyp51 gene or BcatrD gene in WG12 and MX18 were significantly different from each other after treatment with flusilazole. In addition, no point mutations in Cyp51 gene were found in MX18. These data suggest flusilazole is a promising fungicide for resistance management of gray mold and also provided novel insights into understanding the resistance mechanism of flusilazole against plant pathogens.