Botryosphaeria Dothidea Research Articles

A novel mycovirus isolated from the plant-pathogenic fungus Botryosphaeria dothidea.

A novel virus, Botryosphaeria dothidea bipartite mycovirus 1 (BdBMV1), was isolated from the plant-pathogenic fungus Botryosphaeria dothidea strain HNDT1, and the complete nucleotide sequence of its genome was determined. BdBMV1 consists of two genomic segments. The first segment is 1,976bp in length and contains a single open reading frame (ORF) encoding the RNA-dependent RNA polymerase (RdRp) (68.95kDa). The second segment is 1,786bp in length and also contains a single ORF encoding a hypothetical protein of 35.19kDa of unknown function. Based on the sequence of its RdRp, BdBMV1 is phylogenetically related to several other unclassified dsRNA mycoviruses, including Cryphonectria parasitica bipartite mycovirus 1 (CpBV1), and has a distant relationship to members of the family Partitiviridae.

Antagonistic Effects of Delia antiqua (Diptera: Anthomyiidae)-Associated Bacteria Against Four Phytopathogens

Recent studies have revealed multiple roles of insect-associated microbes such as lignin degradation, entomopathogen inhibition, and antibiotic production. These functions improve insect host fitness, and provide a novel source of discovering beneficial microbes for industrial and agricultural production. Previously published research found that in the symbiosis formed by the dipteran pest Delia antiqua (Meigen) (Diptera: Anthomyiidae) and its associated bacteria, the bacteria showed effective inhibition of one fungal entomopathogen, Beauveria bassiana. The antifungal activity of those associated bacteria indicates their potential to be used as biocontrol agents for fungal phytopathogens. In this study, we first isolated and identified bacteria associated with D. antiqua using a culture-dependent method. Second, we tested the antifungal activity of these bacteria against four phytopathogens including Fusarium moniliforme, Botryosphaeria dothidea, and two Fusarium oxysporum strains using the dual-culture method. In total, 74 species belonging to 30 genera, 23 families, eight classes, and four phyla were isolated and identified. Among those bacteria, Ochrobactrum anthropi, Morganella morganii, Arthrobacter sp. 3, and Acinetobacter guillouiae showed significant volatile inhibition activity against F. moniliforme, B. dothidea, and both F. oxysporum, respectively. Moreover, bacteria including Rhodococcus equi, Leucobacter aridicollis, Paenibacillus sp. 3, and Lampropedia sp. showed significant contact inhibition activity against F. moniliforme, B. dothidea, and both F. oxysporum. Our work provides a new source for discovering biocontrol agents against phytopathogens.

Metagenomic-based Approach for the Analysis of Yeast Diversity Associated with Amylase Production in Lai (Durio kutejensis)

This study reported the application of a next generation sequencing (NGS) analysis of yeast diversity in native Indonesian fruit, Durio kutejensis, collected from Borneo, Central Kalimantan. The analysis was designed to observe the microbial consortium associated with solid state fermentation (SSF) for amylase production. Together with the additional data from culture-dependent analysis, we observed the morphological features, molecular characteristics, and amylase concentration produced by each isolate. We performed Solid State Fermentation (SSF) for amylase production and the enzyme activity was then determined using UV-Vis spectrophotometer at 540 nm. Result obtained from metagenomic approach consist of 4 group that fungal species included in the Ascomycota identified as Botryosphaeria dothidea (1.35%), Lasiodiplodia crassispora (17.62%), Aureobasidium pullulans (55.02%), Paraphoma chrysanthemicola (11.38%), Preussia funiculate (1.90%), Sporormiella intermedia (0.82%), Myrothecium gramineum (1.35%), Fusarium oxysporum (6.24%), Fusarium proliferatum (3.25%) and Phialemoniopsis curvata (1.08%). The results of isolation using culturable medium in the form of YMA obtained 40 yeast isolates. A total of 40 representative isolates from durian fruit were screened, two positive amylase isolates based on clear zones formed were DU 4.2 (Candida sorboxylosa) and DU4.22 (Cyberlindnera fabianii) isolates with amylolytic index of DU 4.2 isolates at 0.24 and DU 4.22 at 0.72 with an incubation time of 48 h. The highest amylase enzyme activity was found in isolate DU 4.2 of 31.21 U / mL.

Butylated Hydroxytoluene Induced Resistance Against Botryosphaeria dothidea in Apple Fruit.

Apple ring rot caused by Botryosphaeria dothidea is an important disease in China, which leads to serious economic losses during storage. Plant activators are compounds that induce resistance against pathogen infection and are considered as a promising alternative strategy to traditional chemical treatment. In the present study, butylated hydroxytoluene (BHT), a potential plant activator, was evaluated for its induced resistance against B. dothidea in postharvest apple fruits. The physiological and molecular mechanisms involved in induced resistance were also explored. The results showed that BHT treatment could trigger strong resistance in apple fruits against B. dothidea, and the optimum concentration was 200 μmol L–1 by immersion of fruits. BHT treatment significantly increased the activities of four defensive enzymes and alleviated lipid peroxidation by increasing antioxidant enzyme activities. In addition, salicylic acid (SA) content was enhanced by BHT treatment as well as the expression of three SA biosynthesis-related genes (MdSID2, MdPAD4, and MdEDS1) and two defense genes (MdPR1 and MdPR5). Our results suggest that BHT-conferred resistance against B. dothidea might be mainly through increasing the activities of defense-related enzymes and activating SA signaling pathway, which may provide an alternative strategy to control apple ring rot in postharvest fruits.

Dinâmica populacional de fungos causadores de doenças de tronco em videiras sob a ação de possíveis agentes inibidores

O artigo apresenta contribuições matemáticas para as áreas da agricultura e da fitopatologia, utilizando-se de dados reais e modelando o comportamento de fenômenos físicos. O objetivo do trabalho é modelar o crescimento do Botryosphaeria dothidea em meio de cultura com diferentes componentes, comparando os dados experimentais com o modelo matemático clássico de Verhulst. O experimento foi realizado no Laboratório de Fitopatologia do IFRS - Campus Bento Gonçalves, onde foi preparado o meio de cultura batata-dextrose-ágar (BDA), com diferentes componentes possivelmente inibitórios de crescimento, além de uma testemunha (somente com BDA). O meio de cultura foi alocado em placas de Petri e, em seguida, o fungo Botryosphaeria dothidea foi repicado no centro das placas sobre esse meio. Foram realizadas medições do crescimento fúngico a cada 12 horas. Para o processamento numérico e para a exibição dos gráficos foram utilizados softwares computacionais, tais como o Excel e o Scilab. Por meio de Modelos Matemáticos, que consequentemente refletem em Equações Diferenciais Ordinárias, foi possível programar as simulações numéricas, verificando-se, assim, que a testemunha foi a que apresentou menor erro em relação ao modelo clássico. É plausível afirmar que o crescimento do fungo segue um modelo matemático clássico (Verhulst). Porém, foi possível constatar que os meios de cultura que possuíam possíveis inibidores em sua composição retardaram o crescimento do fungo, indicando que neste tipo de situação o modelo matemático clássico não descreve precisamente a realidade, necessitando ser adaptado.

Candidate Effectors from Botryosphaeria dothidea Suppress Plant Immunity and Contribute to Virulence.

Fungal effectors play important roles in host–pathogen interactions. Botryosphaeria dothidea is an ascomycetous fungus that is responsible for the diseases of hundreds of woody plant species, including apple ring rot, which seriously affects apples worldwide. However, little is known about the effectors of B. dothidea. In this study, we analyzed the B. dothidea genome and predicted 320 candidate effector genes, 124 of which were successfully amplified and cloned. We investigated the effects of these genes on plant cell death in Nicotiana benthamiana while using a transient expression system. Twenty-four hours after initial inoculation with Agrobacterium tumefaciens cells carrying candidate effectors, the infiltrated leaves were challenged with A. tumefaciens cells carrying the BAX gene. In total, 116 candidate effectors completely inhibited, while one partially inhibited, the programmed cell death (PCD) of N. benthamiana induced by BAX, whereas seven candidate effectors had no effect. We then further tested seven candidate effectors able to suppress BAX-triggered PCD (BT-PCD) and found that they all completely inhibited PCD triggered by the elicitors INF1, MKK1, and NPK1. This result suggests that these effectors were activated in order to suppress pathogen-associated molecular pattern-triggered immunity. The signal peptides of these candidate effectors exhibited secretory activity in yeast (pSUC2 vector). Moreover, the respective deletion of Bdo_11198 and Bdo_12090 significantly reduced the virulence of B. dothidea. These results suggest that these effectors play important roles in the interaction of B. dothidea with its hosts.

Genome Wide Transcriptomic Analysis of <i>WRKY</i> Gene Family Response to Biotic Stresses in <i>Malus</i> ×<i>domestica</i>

Apple (Malus × domestica Borkh.) is a perennial woody plant that often suffers from various biological stresses. Many harmful pathogens can infect apple trees and lead to reduced production. We comprehensively identified the WRKY genes in the apple genome and analyzed their expression in response to several biological stressors, including Alternaria alternata, Pythium ultimum, Botryosphaeria dothidea, Erwinia amylovora, Penicillium expansum, Gymnosporangium yamadae, and Apple replant disease. There were 113 MdWRKYs identified in the apple genome. Twenty-two MdWRKYs were differentially expressed in response to at least five pathogens. Promoter sequence analysis showed that these genes carried many defense- and stress-responsive elements, such as MeJA-response elements, salicylic acid-response elements, and W-box elements, in their promoters. Transient expression assays showed that MdWRKY40a and MdWRKY54h played negative roles in defense against B. dothidea infection. WRKY40 and WRKY60 and the MdWKRY33s might play important roles in responding to pathogens and are conserved in some plants. These differentially expressed MdWRKYs might play key roles in the apple response to multiple pathogens.

Pathogenic and Molecular Characterization of Panicle and Shoot Blight Disease (Botryosphaeria dothidea) in Pistachio in the Mediterranean and Southeastern Anatolia Regions

Türkiye, yılda yaklaşık 144 bin ton üretim ile dünyanın üçüncü büyük antepfıstığı üreticisidir. Antepfıstığında tespit edilen fungal hastalıklardan biri de salkım ve sürgün yanıklığına neden olan Botryosphaeria dothidea'dır. Bu patojen son on yılda önemli ve yaygın endofitlerden biri olmuştur. Bu çalışma, 2015 ve 2016 yıllarında Akdeniz ve Güneydoğu Anadolu Bölgesi’nde yer alan Mersin, Gaziantep ve Kilis illerinde antepfıstığı bahçelerinde salkım ve sürgün yanıklık hastalığına neden olan etmenin türünü, yaygınlığını ve şiddetini belirlemek amacıyla yapılmıştır. Çalışma sonucunda, salkım ve sürgün yanıklık hastalığına neden olan etmen hem morfolojik hem de moleküler yöntemler kullanılarak tanılanmış ve Botryosphaeria dothidea olduğu belirlenmiştir. Survey çalışmalarında salkım ve sürgün yanıklık hastalığının en yüksek ortalama yaygınlık oranı ve ortalama hastalık şiddeti sırasıyla, % 71.28 ve % 2.90 olarak Mersin ilinde tespit edilmiştir.

Antimalarials and Phytotoxins from Botryosphaeria dothidea Identified from a Seed of Diseased Torreya taxifolia.

The metabolic pathways in the apicoplast organelle of Plasmodium parasites are similar to those in plastids in plant cells and are suitable targets for malaria drug discovery. Some phytotoxins released by plant pathogenic fungi have been known to target metabolic pathways of the plastid; thus, they may also serve as potential antimalarial drug leads. An EtOAc extract of the broth of the endophyte Botryosphaeria dothidea isolated from a seed collected from a Torreya taxifolia plant with disease symptoms, showed in vitro antimalarial and phytotoxic activities. Bioactivity-guided fractionation of the extract afforded a mixture of two known isomeric phytotoxins, FRT-A and flavipucine (or their enantiomers, sapinopyridione and (-)-flavipucine), and two new unstable γ-lactam alkaloids dothilactaenes A and B. The isomeric mixture of phytotoxins displayed strong phytotoxicity against both a dicot and a monocot and moderate cytotoxicity against a panel of cell lines. Dothilactaene A showed no activity. Dothilactaene B was isolated from the active fraction, which showed moderate in vitro antiplasmodial activity with high selectivity index. In spite of this activity, its instability and various other biological activities shown by related compounds would preclude it from being a viable antimalarial lead.

First Report of Botryosphaeria dothidea Causing Stem Canker on Soybean in China.

In September 2020, widespread stem canker on soybean (Glycine max) was detected in southeastern Jiangsu (Nantong municipality; 120.76° E, 32.23° N) in China. Mature plants, 14 weeks of cultivation, exhibited brown necrotic lesions and dried-up stem. The symptoms were observed in eleven soybean fields, 1.6 ha in total, and approximately 80% of the plants were symptomatic. The symptoms were consistent with those previously reported for stem canker on soybean caused by Diaporthe aspalathi, D. caulivora and D. sojae (Ghimire et al. 2019; Mena et al. 2020). Small pieces, approximately 0.4 cm2 in size, of symptomatic tissue were surface sterilized in 1.5% NaOCl for 1 min, and washed twice with sterile ddH2O. The pathogen was isolated and cultured on potato dextrose agar (PDA), containing chloramphenicol (50 µg/mL), under darkness at 28 ºC for 7 days. Amplification of internal transcribed spacer (ITS), elongation factor 1-α (EF1-α) and β-tubulin (TUB2) genes was performed using ITS1/ITS4, EF1-728F/EF1-986R and Bt2a/Bt2b primers, respectively (Jia et al. 2019). Sequences were submitted to GenBank under accession numbers MW130133 (ITS), MW147481 (EF1-α) and MW147482 (TUB2). Blast search revealed that the amplified sequences had 99.65% (ITS; B. dothidea JZB310202, MN945381), 100% (EF1-α; B. dothidea ZB-77, MH726166) and 99.75% (TUB2; B. dothidea ZB-1, MN642587) matches to multiple B. dothidea strains, whereas all reported Diaporthe strains showed no nucleotide identity to the amplified sequences. Molecular phylogenetic tree was constructed using MEGA7 to confirm the identity of the pathogen. ITS, EF1-α and TUB2 sequences were blasted separately in Muscle (https://www.ebi.ac.uk/Tools/msa/muscle/) and then combined together to make the phylogenetic tree. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model, and the tree with the highest likelihood (-4291.3981) is shown in Figure 1. Diaporthe strains found causing stem canker on soybean, some Phytophthora sojae strains (which also cause dried-up stem on soybean) (Yang et al. 2019), and B. dothidea strains found in China in other hosts were included in the phylogenetic tree. To confirm pathogenicity, a sterilized spatula was used to make wounds (3 mm diameter, 1 mm depth) on the stem of 2-week old soybean plants. Mycelial plugs from a 7 day-old culture on PDA were placed on the wounds and covered with Parafilm. Sterilized PDA plugs were used as control. Inoculated plants were maintained in a growth chamber at 28 °C and 60% relative humidity. Typical stem canker symptoms were observed 5 days after inoculation (Figure 2). Microscope observations showed the presence of septate mycelium, fusiform conidia and round conidiomata, and agreed with those previously reported for the morphology of B. dothidea strains (Phillips et al. 2013). During recent months, B. dothidea was reported to cause stem canker and leave wilt on a number of plant species in China (Huang et al. 2020; Ju et al. 2020; Wang et al. 2020a, 2020b, 2020c), confirming the expansion and host promiscuity of this pathogen. Stem canker on soybean has been thoroughly associated to Diaporthe strains; however, this is the first report of B. dothidea causing this disease. We believe that our results will help to better understand the pathogens affecting soybean production in China.

First report of leaf blight disease of Euonymus japonicus caused by Botryosphaeria dothidea in China

The evergreen shrub species of Euonymus japonicus Thunb. has been widely cultivated as a landscape plant in China. In 2019, leaf blight disease was observed on E. japonicas in Guizhou Province, China, which can cause ultimately tree death. The morphological characteristics and phylogeny of the disease-associated fungus were described and the pathogenicity of this species followed Koch's postulates, confirmed Botryosphaeria dothidea as the causal agent of leaf blight disease of E. japonicas in Guizhou Province, China.

Molecular characterization of a novel mitovirus from the plant‑pathogenic fungus Botryosphaeria dothidea.

Here, a novel mycovirus, Botryosphaeria dothidea mitovirus 1 (BdMV1), was isolated from a phytopathogenic fungus, Botryosphaeria dothidea, and its molecular characteristics were determined. BdMV1 has a genome of 2,667 nt that contains a single large open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF encodes an RNA-dependent RNA polymerase (RdRp) of 727 amino acids with a molecular mass of 81.64 kDa. BLASTp analysis revealed that the RdRp domain of BdMV1 has 39.59% and 39.18% sequence identity to Plasmopara viticola associated mitovirus 43 and Setosphaeria turcica mitovirus 1, respectively. Phylogenetic analysis further suggested that BdMV1 is a new member of the genus Mitovirus within the family Mitoviridae. To the best of our knowledge, this is the first report of a mitovirus in B. dothidea.

CRISPR/Cas9-Mediated Mutagenesis of MdCNGC2 in Apple Callus and VIGS-Mediated Silencing of MdCNGC2 in Fruits Improve Resistance to Botryosphaeria dothidea

Cyclic nucleotide-gated ion channels (CNGCs) have been reported to be involved in multiple plant physiological processes. Their involvement in plant immunity has been studied in several herbal plant species. It remains unclear whether CNGCs in woody plants play a similar role in plant immunity. In the present study, we identified an apple CNGC (designated as MdCNGC2), which is the homolog of Arabidopsis CNGC2. Analysis of tissue distribution revealed that MdCNGC2 was expressed in all tested tissues. Abundant transcripts of MdCNGC2 were observed in leaves and shoot bark. Low expression was observed in fruits and roots. MdCNGC2 expression was induced in apple callus and shoot bark by Botryosphaeria dothidea. The induction of MdCNGC2 was significantly higher in susceptible cultivars “Fuji,” “Ralls Janet,” and “Gala” compared to the resistant cultivar “Jiguan,” suggesting that MdCNGC2 may be a negative regulator of resistance to B. dothidea. MdCNGC2 mutagenesis mediated by gene editing based on the CRISPR/Cas9 system led to constitutive accumulation of SA in apple callus. A culture filtrate of B. dothidea (BCF) induced the expression of several defense-related genes including MdPR1, MdPR2, MdPR4, MdPR5, MdPR8, and MdPR10a. Moreover, the induction of these genes was significantly higher in mdcngc2 mutant (MUT) callus than in wild type (WT) callus. Further analysis showed that the spread of B. dothidea was significantly lower on MUT callus than on WT callus. Knockdown of the MdCNGC2 gene reduced lesions caused by B. dothidea in apple fruits. These results collectively indicate that MdCNGC2 is a negative regulator of resistance to B. dothidea in apple callus.

Improvement of antifungal activity of a culture filtrate of endophytic Bacillus amyloliquefaciens isolated from kiwifruit and its effect on postharvest quality of kiwifruit.

This study reports the optimization of culture conditions and medium components of M9, an endophytic strain of Bacillus amyloliquefaciens, which we had previously isolated from kiwifruit, and evaluation of its effect on kiwifruit postharvest protection against soft rot. The method of one-factor-at-a-time was used to determine the optimum culture conditions, and response surface methodology was applied to optimize the medium constituents. After optimization, a high rate of antifungal activity (73.12% decrease in decay rate) by M9 culture filtrate against the soft rot fungal pathogen, Botryosphaeria dothidea, was obtained. Compared with the initial culture conditions, the antifungal activity of M9 culture filtrate was increased by 19.5%. Furthermore, a postharvest storage experiment on kiwifruit showed that M9 culture filtrate could maintain the quality of stored kiwifruit, delay fruit senescence, and significantly enhance the storability of kiwifruit. Using liquid chromatography-mass spectrometry, we found that the antifungal activity of M9 was associated with the presence of the C12-surfactin A lipopeptide. PRACTICAL APPLICATIONS: Soft rot caused by Botryosphaeria dothidea is one of the most important diseases of kiwifruit, causing postharvest fruit rot. The disease progresses rapidly and is difficult to control, posing a great threat to the kiwifruit industry. In this study, the culture conditions and medium composition for culture of the strain M9 (kiwifruit endophytic Bacillus amyloliquefaciens) were optimized to maximize the inhibition of B. dothidea. A postharvest storage experiment showed that M9 culture filtrate could improve the disease resistance of kiwifruits, delay the senescence of the fruits, and maintain the quality of kiwifruit during storage. Because M9 is a natural endophyte of kiwifruit, the strategy used in this study was both effective and safe. This work will contribute to the exploitation of B. amyloliquefaciens in controlling the pathogens of kiwifruit and the development of safer and more advanced strategies for the postharvest protection of kiwifruits.

First Report of Pomegranate Fruit Rot Caused by Botryosphaeria dothidea in Anhui Province of China

Pomegranate (Punica granatum L.) is an important commercial fruit rich in antioxidants grown worldwide and China has the biggest planting area in the world. In spring 2019, symptoms of fruit rot were observed for the first time in some orchards located in Huaiyuan County of Anhui Province, one of the main pomegranate producing areas in China. Disease symptoms on fruit were characterized by enlarging and merging coarse irregular spots that coalesced and became large decay lesions on the fruit surface. Infected fruits developed internal rot, became discolor and dropped. Aggregated pycnidia were found in a black stroma on fallen mummified fruit. For isolation of the pathogen, more than twenty infected pomegranate fruit samples were collected from an orchard. After surface cleaning with sterile water to clear away some dust and other microbes, dried and disinfected with 70% ethanol for 1 minute, rinsed twice with sterile water, small pieces with diseased and healthy tissue parts were placed on water agar plates (WA). Small piece of every fungal isolate grown on the WA was transferred onto PDA and incubated at 28 °C for 5 days. All fungal colonies on potato dextrose agar (PDA) plates gradually changed from gray to dark grey with olive green back and abundant aerial mycelia, bearing pycnidia with fusiform, hyaline and 16.2 to 20.1× 3.8 to 5.4 μm (n = 50) in size. Twenty isolates with the similar cultural characteristics were stored for further work. The internal transcribed spacer (ITS) region, β-tubulin gene (TUB) and translation elongation factor 1-α (EF 1-α) gene were amplified and sequenced from three typical isolates using the universal primer pairs ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass et al. 1995) and EF1-728F/EF1-986R (Carbone et al. 1999), respectively. The BLAST results of ITS (MN453421), TUB (MN515421), and EF 1-α (MN515422) sequences showed 99.64%, 99.07% and 98.89% identity with the B. dothidea sequences available in the GenBank database (KP708996, MH724212 and KP183206), respectively. To complete Koch's postulates, healthy fresh pomegranate fruits were disinfected with 70% ethanol solution for one minute and rinsed with distilled water. Fruits were allowed to dry and inoculated on the wounded surface using a mycelial plug of 5 mm in diameter excised from the edges of actively growing fungal colonies, fruits were inoculated with PDA plugs served as negative control. Three fruits were used for each treatment and control. These fruits were kept under high humidity (95% RH) using sterile distilled water in artificial incubator and the experiment was repeated twice. Decay lesions resembling natural infections were observed on fruits after 5-7 days of inoculation. Re-isolation of the pathogen, which was also identified based on the above morphological and molecular analysis as B. dothidea, produced the same fungus as the one used for inoculating the fruit, thus completing the Koch's postulates. Therefore, the pathogen of this pomegranate fruit rot in China is B. dothidea. Botryosphaeria pomegranate rot caused by B. dothidea in Florida has been reported previously and was hypothesized that this fungus could be transmitted by leaf-footed bug (Leptoglossus clypealis, Michailides, 2014). To our knowledge, this is the first report of B. dothidea causing fruit rot on pomegranate in China.

Diversity, distribution and host association of Botryosphaeriaceae species causing oak decline across different forest ecosystems in Algeria

Botryosphaeriaceous fungi associated with holm (Q. ilex) and cork (Q. suber) oak trees exhibiting dieback symptoms and cankers in Algeria, were sampled in twelve regions with different ecological conditions. Based on phylogenetic analyses of ITS and tef1-α loci, 13 species were identified. Three species (Diplodia corticola, Dothiorella iberica and Botryosphaeria dothidea) were found to be common to both oak species, while Doth. sarmentorum, D. mutila, Neofusicoccum vitifusiforme, D. insularis, N. stellenboschiana, Doth. plurivora, D. seriata, D. rosacearum, N. parvum and N. terminaliae were found only on one of the hosts. All the Botryosphaeriaceae taxa inoculated on oak shoots produced brown vascular discolouration, and were shown to be pathogenic on both oak species. Diplodia corticola was the most widespread and frequent species showing the highest aggressiveness towards both hosts. Neofusicoccum stellenboschiana, D. insularis and Doth. plurivora are recorded for the first time on Q. suber and Q. ilex. In addition to altitude, the environmental factors temperature and drought appeared to be influential variables that could describe the occurrence and the geographic distribution of these fungal species.

Antifungal activity of rapamycin on Botryosphaeria dothidea and its effect against Chinese hickory canker.

The trunk canker caused by Botryosphaeria dothidea is a devastating disease for Chinese hickory (Carya cathayensis) in China. Chemical fungicides are commonly and repeatedly applied to control the disease; however, fungicide application raises major environmental and food safety issues. Ecofriendly biocontrol alternatives were urgently needed. Herein, the antifungal activity of a natural secondary metabolite, rapamycin, against B. dothidea and the effect of a rapamycin-producing Streptomyces hygroscopicus on C. cathayensis canker were investigated. The 50% effective concentrations (EC50 ) of rapamycin against mycelial growth and spore germination on the potato dextrose agar were 5.1 × 10-3 and 5.5× 10-2 μg mL-1 , respectively, which were much lower than the EC50 values of thiophanate-methyl. The hyphae of B. dothidea exhibited premature aging and wrinkling after treatment with rapamycin at 5.0 × 10-3 μg mL-1 . A rapamycin-producing bacterium S. hygroscopicus LYJ637 was batch produced and formulated in a carboxymethylcellulose/poly (vinyl alcohol) (CMC/PVA) blend and used for testing the efficiency of the bacterium in controlling Botryosphaeria canker in C. cathayensis. S. hygroscopicus exhibited high stability in the CMC/PVA blend. Results of a 3-year field experiment suggested that rapamycin formation reduced the occurrence of both developed cankers and new cankers, with an efficacy comparable to the treatment with thiophanate-methyl. The rapamycin-producing S. hygroscopicus LYJ637 carried in a CMC/PVA blend prevented effectively Botryosphaeria canker on Chinese hickory, which provides an alternative approach to chemical control strategies.

Effects of ultrasound on submerged fermentation for producing antioxidant metabolites from Botryosphaeria dothidea

Antioxidant compounds were produced from submerged fermentation using Botryosphaeria dothidea. Sonication times (1–15 min) and agitations (0–150 rpm) applied to different fermentation phases (2–7 days) were evaluated for their effect on the broth antioxidant activity. In approximately 70% of assays performed with ultrasound, the antioxidant activity of the broth containing exocellular metabolites was higher than the control (without sonication), reaching a maximum value of 96%. The solid fraction (biomass after supernatant removal) corresponding to the best assay of antioxidant activity was submitted to extractions with water, ethanol or ethyl acetate to recover intracellular metabolites. The yields of extracts were 3.7 ± 2.6 wt %, 20.6 ± 3.9 wt % and 32.0 ± 4.2 wt % for ethyl acetate, ethanol and water, respectively. The use of ethanol could provide a larger number of compounds such as pentadecanoic acid, ergosta-5,8,22-trien-3-ol, and 1,4-diaza-2,5-dioxobicyclo [4.3.0] nonane. Both the supernatant and solid fractions revealed some bioactive compounds with promising antioxidant activity with the potential to replace some synthetic antioxidants.

Biological control of poplar anthracnose caused by Colletotrichum gloeosporioides (Penz.) Penz. & Sacc.

Poplar anthracnose is one of the most serious diseases caused by the fungal pathogen Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. Biocontrol is an efficient green way for the disease control, and numerous researches have focused on exploring the potential biocontrol bacteria strains against C. gloeosporioides. In this study, antifungal activities against C. gloeosporioides of 108 rhizosphere soil isolates from healthy polar plants were investigated in vitro by the dual culture assay. The results suggested that strain ZSH-1 showed the highest level of antifungal activity, as it inhibited C. gloeosporioides at a distance of 10.00 mm. Based on the morphological, physiological-biochemical characteristics, and phylogeny analysis, strain ZSH-1 was identified as Bacillus subtilis. The sterile culture filtrate, crude protein, and crude lipopeptide extracts from the culture filtrate, and volatile compound(s) of ZSH-1 displayed a strong antagonism towards 7 fungal phytopathogens (C. gloeosporioides, Fusarium oxysporum, Alternaria tenuissima, Cytospora chrysosperma, Botryosphaeria dothidea, Mucor sp., and Absidia sp.), with inhibition rates ranging from 44.0 to 89.1%, 26.7 to 85.4%, 11.6 to 89.7%, and 7.8 to 63.2%, respectively. Moreover, ZSH-1 exhibited cell wall-degrading traits by producing 3 lytic enzymes (cellulose, β-1,3-glucanase, and protease). Finally, the greenhouse studies also revealed that strain ZSH-1 had a 47.6% (12 days) efficacy in controlling poplar anthracnose when compared with the control. In concluding, obtained results demonstrate the potential biocontrol effect of B. subtilis ZSH-1, and it can be used as a promising biocontrol agent against poplar anthracnose and other fungal phytopathogens.

Pepper growth promotion and Fusarium wilt biocontrol by Trichoderma hamatum MHT1134

ABSTRACT Pepper wilt, caused by Fusarium oxysporum, is an important disease of pepper in many regions of the world. A phosphate solubilising Trichoderma strain MHT1134 was selected from 76 isolates from the pepper rhizosphere as a potential biological control agent against wilt disease caused by F. oxysporum. The inhibitory rate of MHT1134 against F. oxysporum was 81.80%. MHT1134 was identified as Trichoderma hamatum based on morphological features, ITS rDNA and tef-1α sequences. It showed antagonistic effects against a number of fungal pathogens, including Phytophthora capsici, Colletotrichum capsici, Verticillium dahliae, Verticillium albo-atrum, Botryosphaeria dothidea, Pyricularia grisea and Phoma lingam. The fungistatic capability of strain MHT1134 was evaluated, and its sterilised and non-sterilised fermentation broths were effectively suppressed pathogen’s hyphal growth, with inhibitory effects reaching 50.71% and 74.01%, respectively. Moreover, T. hamatum MHT1134 controlled pepper wilt at a 60.61% efficiency level, and applications significantly increased plant heights, root lengths, fresh and dry weights of pepper in pot experiments. The field experiments showed that strain MHT1134 inhibited pepper Fusarium wilt, with a control effect that was only slightly less than that of the chemical hymexazol, and significantly increased pepper yield. Thus, MHT1134 is proposed as a potential biocontrol fungus and further studies will focus on its biocontrol mechanisms.