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In August 2016, tomato plants grown during a hot, wet summer with heavy soil flooding, displaying symptoms of wilting, dead plant, root rot with crown and stem rot, at Beni Suef and Fayoum governorates were examined. A number of 16 fungal isolates were isolated from tomato plants displaying the above symptoms. These isolates were classified as belonging to six species, namely: Alternaria solani, Chaetomium globosum, Fusarium solani, Fusarium oxysporum, Pythium spp. and Rhizoctonia solani. Isolates of Pythium spp. were prevalent and were found to be more pathogenic than the other fungal isolates. This species causes damping-off, root rot, sudden death, stem rot and fruit rot. The pathogen was identified as Pythium aphanidermatum based on morphological, cultural, and molecular characteristics. Biogenic silver nanoparticles (AgNPs) were produced using the F. oxysporum strain and characterized by transmission electron microscopy (TEM). The size of these spherical particles ranged from 10 to 30 nm. In vitro, biogenic AgNPs showed antifungal activity against P. aphanidermatum. In greenhouse and field experiments, AgNPs treatment significantly reduced the incidence of dead tomato plants due to root rot caused by P. aphanidermatum compared to the control. All of the investigated treatments were effective and the treatment of root dipping plus soil drenching was the most effective. To the best of our knowledge, this study describes P. aphanidermatum on tomato in Egypt for the first time. Also, biogenic AgNPs could be used for controlling root rot disease caused by this pathogen.

The loss of fresh produces owing to the microbial infestation is a major challenge to the global food industry. The drastic food loss caused mainly by the fungal attack demands the need for development of active packaging materials with antimicrobial properties. Many studies have already been reported on the applications of polymers like polyvinyl alcohol (PVA) engineered with antimicrobial components as active antifungal packaging materials. In the current study, material properties of PVA alone, PVA incorporated with chitosan nanoparticles (PCS), clove oil (PCO), and their combination (PCSCO) have been studied for its microbial barrier and antifungal properties. All the developed films were characterised by the XRD and FTIR analysis, which confirmed the molecular interactions among the individual components of the nanocomposite. At the same time, the bionanocomposite PCSCO was found to have low moisture content and film solubility indicating its suitability for the modified atmosphere packaging applications. In addition, the presence of chitosan nanoparticles and clove oil was found to provide the microbial barrier properties to the PCS, PCO, and PCSCO films. The PCSCO film was further demonstrated to have superior antifungal activity against the selected Pythium aphanidermatum. The results of the study indicate the potential application of developed nanocomposite film as a promising antifungal packaging material.

Plant growth-promoting rhizobacteria benefit plants by producing phytohormones and acquiring vital nutrients from soil. They also form a shield against potential phytopathogens with the production of antimicrobial compounds such as bacteriocin, biosurfactants etc. In the present study, among various rhizobacteria isolated from ginger rhizosphere, the isolate identified as Bacillus altitudinis VS7 that showed a potent biosurfactant producing ability in the preliminary tests was selected for the further study. Compositional analyses indicated that the biosurfactant produced by VS7 was a surfactin like compound, a cyclic lipopeptide. In both microtitre plate and mycelium growth inhibition studies, the biosurfactant showed dose dependent antagonistic activities against Pythium aphanidermatum that causes soft rot in ginger. Although there are reports of biocontrol activity of various rhizobacteria against soft rot in ginger, this is the firstinformation regarding biocontrol activity of a biosurfactant produced by the rhizobacterium B. altitudinis VS7 against P. aphanidermatum.

Pythium aphanidermatum is the predominant species causing Pythium root rot of commercially grown poinsettia (Euphorbia pulcherrima Willd. ex Kotzch) in North Carolina. Pythium root rot is managed primarily through a combination of sanitation practices and preventative fungicide applications of mefenoxam or etridiazole. Insensitivity to mefenoxam is common but growers continue to rely on it from lack of inexpensive and efficacious alternatives. This research was conducted to identify alternative fungicides for Pythium root rot control and to evaluate their efficacy on poinsettia cultivars with varying levels of partial resistance. Greenhouse studies were conducted to assess efficacy of fungicide treatments in seven poinsettia cultivars inoculated with a mefenoxam-sensitive isolate of P. aphanidermatum. One study examined control with a single fungicide drench made at transplant and a second study examined repeat fungicide applications made throughout the experiment. Treatments containing etridiazole, mefenoxam, fenamidone, and cyazofamid provided control of Pythium root rot across all cultivars in both experiments whereas Fosetyl-al, potassium phosphite, and Trichoderma spp. failed to offer satisfactory control. Azoxystrobin, pyraclostrobin, and propamocarb reduced disease on some cultivars but failed to control Pythium root rot on highly susceptible cultivars. Four isolates of P. aphanidermatum cultured from plants growing in commercial greenhouses were evaluated for in vitro sensitivity to fungicides labeled for Pythium root rot control at four rates. Etridiazole, fosetyl-al, and potassium phosphite completely inhibited mycelial growth, whereas isolates varied in response to mefenoxam, cyazofamid, propamocarb, fenamidone, azoxystrobin, and pyraclostrobin in vitro. Twenty-one additional isolates then were evaluated at label rates of these fungicides. Seven isolates were insensitive to label rates of all three quinone outside inhibitors and one isolate was insensitive to the quinone outside inhibitors and mefenoxam. These results provide guidelines for selecting fungicides to maximize control of Pythium root rot on poinsettia cultivars.

Background Succinate dehydrogenase inhibitors (SDHIs) play an increasingly important role in controlling plant diseases. However, the similar structures of SDHIs result in rapid development of cross-resistance development and a clear bottleneck of poor activity against oomycetes. Therefore, the need to seek new SDHI fungicides with novel structure is urgent. Results Innovative pyrazolyl oxime ethers were designed by replacing amide with oxime ether based on the SDH structure, and 19 pairs of Z- and E-isomers were efficiently prepared for the discovery of SDHI compounds with novel bridge. Their biological activities against four fungi and two oomycetes were evaluated, and substantial differences were observed between Z- and E- isomer of the title compounds. Furthermore, most of these compounds exhibited remarkable activities against Rhizoctonia solani with EC50 values of less than 10 mg/L in vitro, and bioassay in vivo further confirmed that E-I-6 exhibited good protective efficacy (76.12%) at 200 mg/L. In addition, Z-I-9 provided better activity against the oomycetes Pythium aphanidermatum and Phytophthora capsici (EC50 = 1.56 mg/L and 0.93 mg/L) than those of boscalid. Moreover, E-I-9 exhibited excellent SDH inhibition (IC50 = 0.21 mg/L) thanks to its good binding ability to the SDH by hydrogen-bonding interactions, π-cation interaction and hydrophobic interactions. Conclusion Novel pyrazolyl oxime ethers have the potential as SDHI compounds for future development, and the strategy of replacing amide bond with oxime ether may offer an alternative option in SDHI fungicide discovery. This article is protected by copyright. All rights reserved.

Biological control of plant pathogens is considered as one of the green and effective technologies using beneficial microorganisms or microbial secondary metabolites against plant diseases, and so microbial natural products have played important roles in the research and development of new and green agrochemicals. To explore the potential applications for natural γ-lactam alkaloids and their derivatives, 26 γ-lactams that have flexible substituent patterns were synthesized and characterized, and their in vitro antifungal activities against eight kinds of plant pathogens belonging to oomycetes, basidiomycetes, and deuteromycetes were fully evaluated. In addition, the high potential compounds were further tested using an in vivo assay against Phytophthora blight of pepper to verify a practical application for controlling oomycete diseases. The potential modes of action for compound D1 against Phytophthora capsici were also investigated using microscopic technology (optical microscopy, scanning electron microscopy, and transmission electron microscopy) and label-free quantitative proteomics analysis. The results demonstrated that compound D1 may be a potential novel fungicidal agent against oomycete diseases (EC50 = 4.9748 μg·mL-1 for P. capsici and EC50 = 5.1602 μg·mL-1 for Pythium aphanidermatum) that can act on steroid biosynthesis, which can provide a certain theoretical basis for the development of natural lactam derivatives as potential antifungal agents.

Cladosporium omanense has been recently described as a novel endophytic fungus from Zygophyllum coccineum in the desert habitat of Oman. The ability of this fungus to suppress Pythium aphanidermatu...

Pythium species cause root and stem rot in watermelon (Citrullus lanatus), but cucurbit rootstocks used to graft watermelon have not been evaluated for resistance. P. aphanidermatum and P. myriotylum were inoculated onto 15 nongrafted watermelon, citron (Citrullus amarus), bottle gourd (Lagenaria siceraria), and interspecific hybrid squash (Cucurbita maxima × C. moschata) cultivars in a growth chamber. Watermelon was more susceptible than bottle gourd and interspecific hybrid squash at 20 and 30°C. Twenty-one cultivars were inoculated in a field with an equal blend of both Pythium species. Interspecific hybrid squash was less susceptible than bottle gourd and watermelon in 2018 and 2019. Seedless watermelon cultivar Tri-X 313 was grafted to one citron, one bottle gourd, and three interspecific hybrid squash rootstocks. Plants were inoculated in the field as described. Grafting to interspecific hybrid squash rootstocks reduced disease incidence compared with nongrafted controls in 2018 and 2019. Mefenoxam and propamocarb applied at transplanting did not affect disease compared with non-fungicide-treated plots. Grafting to interspecific hybrid squash Camelforce significantly increased total and marketable fruit numbers and total weight in 2019 compared with the nongrafted control. In summary, interspecific hybrid squash was consistently resistant to Pythium, demonstrating resistance and utility in watermelon grafting.

Background Lipopeptides from the Bacillus spp. possess an excellent spectrum of antimicrobial properties which make them suitable candidates to be explored for the food, agricultural, pharmaceutical and biotechnological applications. As the low yield of the lipopeptides limits their applications, methods to enhance their production are highly significant. Results In this study, extracts prepared from endophytic Bacillus sp. Fcl1 cultured in the presence of various supplements were screened for antifungal activity against Pythium aphanidermatum. From the results, the supplementation of carbon sources and zinc oxide nanoparticles (ZnONPs) was found to have an enhancement effect on the antifungal activity of Bacillus sp. Fcl1. Among these, the highest antifungal activity (73.2%) could be observed for the Fcl1 sample cultured with 5 mg L-1 of ZnONP supplementation. The growth of Fcl1 in the presence of ZnONPs also indicated its compatibility with the nano-supplement in the concentration range used. By liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) analysis, the synthesis of increased numbers of lipopeptide surfactin derivatives could be identified from the extracts of Fcl1 prepared from the carbon sources and ZnONP-supplemented cultures. In addition to the surfactin derivatives, the presence of another lipopeptide iturin was also detected from the extracts of Fcl1 cultured with ZnONPs. Conclusion ZnONP supplementation was found to enhance antifungal activity and lipopeptide production in the Bacillus sp. Fcl1. The use of nanoparticles to enhance the antifungal mechanisms of Fcl1 as observed in the study provides novel insights to explore its applications for sustainable agricultural productivity.

Fruit of Allanblackia floribunda is an active ingredient in the pharmaceutical industry. Fruit production in the species is hindered by incidence of pathogenic fungi leading to economic loss. This study was conducted to investigate micro-fungi species associated with leaf spot of A. floribunda. Field surveys were carried out in natural stands containing matured A. floribunda trees located at Owu Ikija, Ogun State (6.80°N, 4.03°E) and Benin, Edo State (6.29°N and 5.58°E) in Southern Nigeria. Diseased leaf samples were collected during wet and dry seasons. Pure cultures of fungal isolates obtained from the leaf samples were examined to determine their cultural and morphological characteristics. Percentage incidence of micro-fungi in each location was estimated. Leaves of healthy seedlings were sprayed with 104 conidial/ml spore concentration of fungal isolates to determine their pathogenicity. Fourteen fungal species were isolated from leaves of A. floribunda across the two sites. Aspergillus spp., Macrophomina phaseolina, Penicillium species, Pestalotia palmarum, Rhizopus nigricans and Trichoderma pseudokoningii were isolated from both sites during both seasons. Fusarium oxysporum, Lasiodiplodia theobromae, Penicillium javanicum and Pythium aphanidermatum were present at Owu Ikija while Colletotrichum capsici, C. coccodes and Curvularia lunata were present at Benin with fungal incidence of 12.5, 12.5 and 4.17%, respectively. P. palmarum had modal fungal incidence (35.29%) at Benin followed by T. pseudokoningii at Owu-Ikija with frequency value of 18.75 and 17.54%, respectively. P. palmarum was the most prevalent out of all micro-fungi species associated with A. floribunda in all locations. Pathogenicity test was negative for all tested isolates, variety of micro-fungi are associated with A. floribunda. Key words: Allanblackia floribunda, fruit, fungal isolates, leaf samples, pathogenicity.

This study was conducted to investigate the antagonistic activity of endophytic and rhizosphere fungi isolated from a medicinal plant, Sesuvium portulacastrum, against Pythium aphanidermatum, the cause of damping off of cucumber. A total of 40 endophytic and 19 rhizosphere fungi were isolated from S. portulacastrum. Three endophytic isolates and two rhizosphere isolates gave >50% suppression of P. aphanidermatum in the in vitro dual-culture tests. Scanning electron microscopic studies at the inhibition zone showed hyphae wall damage and abnormal mycelial growth of the genus Pythium. Molecular analysis identified the antagonistic endophytes as Aspergillus insulicola (isolate A435), A. insulicola (A419), and Aspergillus melleus (A412) and the rhizosphere antagonists as Aspergillus terreus (A213) and Aspergillus luchuensis (A116). Except for A116, the culture filtrates of the other antagonists significantly increased the electrolyte leakage from Pythium mycelia, whereas ethyl acetate extracts of A435, A412, and A213 showed significant growth suppression. All five antagonists were able to produce varying amounts of cellulase and β-glucanase enzymes. However, A435, A412, and A213 showed significantly higher cellulase activity, whereas A435 and A116 showed the highest β-glucanase activity. Controlled glasshouse growth experiments showed that isolates A435 and A116 resulted in up to 70% control of damping off, whereas isolates A412 and A213 showed 30 to 40% damping-off control. The antagonists A435, A116, and A213 also contributed to increased cucumber shoot length as well as shoot and root dry mass. The synergetic effects of metabolites and hydrolytic enzymes could be the reason for the variation between isolates in the antagonistic activity and cucumber growth promotion. This study reports for the first time A. insulicola, A. melleus, and A. luchuensis as potential biocontrol agents against P. aphanidermatum-induced damping off of cucumber.

The soilborne oomycete pathogen Pythium aphanidermatum causes damping-off and root rot in a wide range of economically important crops and vegetables. Effective P. aphanidermatum disease management...

Fungal wilt and root rot diseases affecting tomato have become prevalent in China in recent years and have caused considerable damage. In 2016 to 2018, symptoms of putative wilt and root rot diseases were observed in several locations in tomato cultivars with resistance to Fusarium oxysporum f. sp. lycopersici races 1 and 2. The objective of this study was to identify the causative agents of wilt and root rot of tomato in China and provide a basis for disease prevention and resistance breeding programs. Based on DNA sequence analyses of the internal transcribed spacer (ITS) region, 91 isolates from the roots of tomato plants showing symptoms of wilt and root rot were identified, including F. oxysporum (64 isolates), Fusarium solani (11 isolates), Fusarium proliferatum (2 isolates), Fusarium graminearum (2 isolates), Fusarium equiseti (1 isolate), Pythium aphanidermatum (6 isolates), Ascomycota sp. (2 isolates), and Plectosphaerella cucumerina (3 isolates). F. oxysporum accounted for 70.33% of the isolates obtained. In this case, using PCR-based methods for differentiation of F. oxysporum, we identified several formae speciales and races of F. oxysporum: 7 isolates were identified as F. oxysporum f. sp. lycopersici race 1, 2 isolates as F. oxysporum f. sp. lycopersici race 2, 35 isolates as F. oxysporum f. sp. lycopersici race 3, and 13 isolates as F. oxysporum f. sp. radicis-lycopersici. Pathogenicity tests revealed 55 isolates of tomato wilt and root rot pathogens to be virulent. This study demonstrated that F. oxysporum f. sp. lycopersici race 3 was the most widespread and highly virulent race among these tomato pathogens in China, followed by F. oxysporum f. sp. radicis-lycopersici. Therefore, the development of resistant varieties of tomato against F. oxysporum f. sp. lycopersici race 3 and F. oxysporum f. sp. radicis-lycopersici would aid efforts to develop effective disease management strategies.

This study was conducted to investigate the presence of native antagonistic bacteria from compost and to evaluate their ability to suppress Pythium and Fusarium-induced damping-off of cucumber. Sev...

Aims Diseases caused by pathogenic fungi was a major constrain in increasing productivity and improving quality of Panax notoginseng. The aim of this research was to evaluate the inhibitory activity of essential oils (EOs) from Asteraceae family, Chrysanthemum indicum and Laggera pterodonta, against pathogenic fungi of P. notoginseng. Methods and results The antifungal activity was investigated using multiple methods, disclosing that the EOs from C. indicum and L. pterodonta are active against hypha growth of different fungi but with different degrees of potency. Checkerboard testing indicated that the combination of EOs with hymexazol had synergistic effect against Pythium aphanidermatum, and exhibited additive effects against bulk of targeted pathogenic fungi. Besides, we found that the baseline sensitivity of Fusarium oxysporum to L. pterodonta EOs was higher than those of C. indicum by means of mycelium growth rate method. Finally, the practicability of those EOs as plant pesticide was confirmed by in vivo model showing that EOs can significantly inhibit the occurrence of root rot of P. notoginseng caused by F. oxysporum. Conclusion Those studies suggest that the EOs from C. indicum and L. pterodonta had the potential to develop into new pollution-free pesticides for the protection of precious Chinese herbal medicines. Significance and impact of the study This study provided a new way of biological control for overcoming the frequent diseases occurrence of P. notoginseng.

Background Pythium, soil-borne plant pathogens, are in the class Oomycetes. They are not true fungi, but are related to diatom and algae. There are two human pathogens including P. insidiosum and P. aphanidermatum. To date, only one case of pythiosis caused by P. aphanidermatum has been reported. We present herein the first case of P. aphanidermatum vascular pythiosis in Asia. Case presentation A 47-year-old Thai woman, living in North Thailand, with ß thalassemia/hemoglobin E presented with acute recurrent arterial insufficiency of both legs. Emergent embolectomy with clot removal was performed. The pathology of the clot exhibited noncaseous granulomatous inflammation with many fungal hyphal elements. PCR identified P. aphanidermatum with 100% identity. Final diagnosis is vascular pythiosis. Unfortunately, the patient eventually expired after treatment with itraconazole, terbinafine, azithromycin, and doxycycline. Conclusions To date, only one case of pythiosis caused by P. aphanidermatum has been reported. We present herein the first case of P. aphanidermatum vascular pythiosis in Asia.

Plants recognize a variety of external signals and induce appropriate mechanisms to increase their tolerance to biotic and abiotic stresses. Precise recognition of attacking pathogens and induction of effective resistance mechanisms are critical functions for plant survival. Some molecular patterns unique to a certain group of microbes (MAMPs, microbe-associated molecular patterns) are sensed by plant cells as non-self molecules via pattern recognition receptors. While a variety of MAMPs of bacterial and fungal origin have been identified, reports on MAMPs of oomycete pathogens are relatively limited. This study aimed to identify unique MAMP elicitors from the oomycete pathogen Phytophthora infestans, the causal agent of potato late blight. Using reactive oxygen species (ROS) production and phytoalexin production in potato as markers for the purification of oomycete elicitors, we identified two structurally different groups of elicitors, namely ceramides and diacylglycerols. P. infestans ceramide (Pi-Cer) elicitors induced ROS production, while diacylglycerol (Pi-DAG) elicitors, containing eicosapentaenoic acid (EPA) as a substructure, induced the formation of phytoalexins in potato. Pi-Cer and Pi-DAG are also contained in the mycelia of another oomycete pathogen Pythium aphanidermatum, indicating that they are MAMPs of oomycetes. When Arabidopsis was treated with Pi-Cer and EPA, partially overlapping but different sets of genes were induced. Furthermore, simultaneous treatment with Pi-Cer and EPA did not have a cumulative effect on induced genes, but rather the expression of some genes induced by EPA was attenuated by the co-treatment with Pi-Cer. These results indicate that plants may combine the signals from simultaneously recognized MAMP elicitors to specifically adapt the defense response to a particular pathogen.

Damping-off, root rot and necrotic lesions were observed on sugar beet (Beta vulgaris) seedlings in the Kafrelsheikh Governorate, in the northern part of the Nile River delta, Egypt. Pythium aphanidermatum was identified as the causal agent using the morphology of the asexual and sexual morphs and molecular studies. This is the first record of P. aphanidermatum on sugar beet in Egypt.

(1) Background: biological way is one of the most ecofriendly and safe strategies for nanomaterials synthesis. So, biosynthesis-green method was used for the preparation of Zn(II) complex (in the Nano scale) from the reaction of the schiff base ligand 2,2′-((1E,1′E)-(1,2-phenylenebis (azanylylidene)), bis(methanylylidene))bis(4-bromophenol), and Zn(II)sulphate. The biogenic ZnNP-T was characterized by different methods. Our purpose was to evaluate the ability of biosynthesis-green method for the preparation of Zn(II) complex as an antifungal agent against diseases from fungal species. (2) Methods: in this work, isolates of Pythium aphanidermatum and Trichderma longibrachiatum were obtained, and Trichderma longibrachiatum was used to prepare nano metal complex. We tested the pathogenicity of nano metal complex against seedling and germination of seeds, and we evaluated the effectiveness of ZnNP-T for growth promotion of Vicia feba in early stage and inhibitory activity against Pythium aphanidermatum. (3) Results: antagonistic activity of ZnNP-T was tested in vitro against Pythium aphanidermatum, and then the growth rates of Vicia faba were determined. The obtained data revealed that mycelial growth of pathogenic fungus was inhibited about 73.8% at 20 ppm. In addition, improved the total biomass of Vicia faba in the presence of P. aphanidermatum. All concentration of ZnNP-T positively affected root weight of Vicia faba seedlings, and positively affected shoot weight. Root and shoot lengths were affected by using 20 ppm of ZnNP-T with up to 180 and 96.5 mm of shoot and root length compared to that of the control, while germination percentage was significantly enhanced with up to 100% increase after 72 h of germination. (4) Conclusion: one of the modern challenges in vegetable or fruit production is to enhance seed germination and to grow healthy plants with strong root system. In future, there should be a focus on using of biogenic Zinc nano-complex as plant growth promoter agents.

Trichoderma is the most commonly used fungal biocontrol agent throughout the world. In the present study, various Trichoderma isolates were isolated from different vegetable fields. In the isolated microflora, the colony edges varied from wavy to smooth. The mycelial forms were predominantly floccose with hyaline color and conidiophores among all the strains were highly branched. Based on morphological attributes, all the isolates were identified as Trichoderma harzianum. The molecular identification using multilocus sequencing ITS, rpb2 and tef1α, genes further confirmed the morphological identification. The average chitinase activity varied from 1.13 units/mL to 3.38 units/mL among the various isolates, which increased linearly with temperature from 15 to 30 °C. There was an amplified production in the chitinase production in the presence of Mg+ and Ca2+ and Na+ metal ions, but the presence of certain ions was found to cause the down-regulated chitinase activity, i.e., Zn2+, Hg2+, Fe2+, Ag+ and K+. All the chitinase producing Trichoderma isolates inhibited the growth of tested pathogens viz., Dematophora necatrix, Fusarium solani, Fusarium oxysporum and Pythium aphanidermatum at 25% culture-free filtrate concentration under in vitro conditions. Also, under in vivo conditions, the lowest wilt incidence and highest disease control on Fusarium oxysporum was observed in isolate BT4 with mean wilt incidence and disease control of 21% and 48%, respectively. The Trichoderma harzianum identified in this study will be further used in formulation development for the management of diseases under field conditions.

Ethaboxam is a benzamide antioomycete chemical (oomicide) used in corn and soybean seed treatments. Benzamides are hypothesized to bind to β-tubulin, thus disrupting microtubule assembly. Recently, there have been reports of corn- and soybean-associated oomycetes that are insensitive to ethaboxam despite never having been exposed. Here, we investigate the evolutionary history and molecular mechanism of ethaboxam insensitivity. We tested the sensitivity of 194 isolates representing 83 species across four oomycete genera in the Peronosporalean lineage that were never exposed to ethaboxam. In all, 84% of isolates were sensitive to ethaboxam (effective concentration to reduce optical density at 600 nm by 50% when compared with the nonamended control [EC50] < 5 μg ml−1), whereas 16% were insensitive (EC50 > 11 μg ml−1). Of the insensitive isolates, two different transversion mutations were present in the 239th codon in β-tubulin within three monophyletic groups of Pythium spp. The transversion mutations lead to the same amino acid change from an ancestral cysteine to serine (C239S), which coincides with ethaboxam insensitivity. In a treated soybean seed virulence assay, disease severity was not reduced on ethaboxam-treated seed for an isolate of Pythium aphanidermatum containing a S239 but was reduced for an isolate of P. irregulare containing a C239. We queried publicly available β-tubulin sequences from other oomycetes in the Peronosporalean lineage to search for C239S mutations from other species not represented in our collection. This search resulted in other taxa that were either homozygous or heterozygous for C239S, including all available species within the genus Peronospora. Evidence presented herein supports the hypothesis that the convergent evolution of C239S within Peronosporalean oomycetes occurred without selection from ethaboxam yet confers insensitivity. We propose several evolutionary hypotheses for the repeated evolution of the C239S mutation.

Bu çalışma, Sakarya, Kocaeli, Yalova, Bursa, İstanbul, Kırklareli ve Edirne illerine bağlı, odunsu bitkilerin üretiminin yapıldığı, 13 adet devlet ve 3 adet özel fidanlıkta, fungus ve su küflerinin varlığını belirlemek amacıyla gerçekleştirilmiştir. Çalışmada, adı geçen fidanlıklarda yetiştirilmekte olan orman ağacı fidanları incelenmiş, geriye doğru ölüm ve kuruma başta olmak üzere çeşitli hastalık semptomları gösteren fidanlar toprakları ile birlikte örneklenmiştir. Laboratuvar çalışmaları; mikroskobik incelemeler ve hastalıklı dokularından fungus, tüplü ve çıplak köklü fidanlara ait toprak örneklerinden de Pythiaceous türlerin izolasyonlarını kapsamıştır. Fidanlıklardan 227 adet fidan ve 79 adet toprak örneği alınmış, bunlardan, 145 adet fungal ve 57 adet Pythiaceous izolatı elde edilmiştir. İzolatlar morfolojik özelliklerine göre gruplandırılmış ve bu gruplar içerisinden seçilen temsili izolatların DNA ekstraksiyonu gerçekleştirildikten sonra, ITS bölgelerinin amplifikasyonu gerçekleştirilerek, dizilemeye gönderilmiştir. Çalışma sonucunda örnekler üzerinde tespit edilen funguslar arasında, dünya çapında kavakların bilinen en yaygın patojenlerinden biri olan Marssonina brunnea, pas hastalığı etmeni, Melampsora allii-populina ve çamlarda kırmızı bant hastalığı olarak bilinen Dothistroma septosporum yer almıştır. Bitki dokularından ve topraktan izole edilen Fusarium solani, Fusarium oxysporum, Pythium aphanidermatum, Pythium irregulare, Pythium ultimum, Phytopythium vexans ve Phytophthora syringae yaygın ve önemli fungus ve Pythiaceous türler olarak ortaya koyulmuştur. Bu araştırma kapsamında gerçekleştirilen sürveyler ve laboratuvar çalışmaları, geniş yapraklı türlerin, iğne yapraklılara, devlet fidanlıklarının da özel fidanlıklara kıyasla daha fazla fungal ve Pythiaceous türlere konukçuluk ettiğini göstermektedir. Elde edilen fungal ve Pythiaceous türlerin miktarı ve çeşitliliği, bu mikroorganizmaların, bir fidanlıktan diğerine ve hatta ağaçlandırma sahalarına taşınabilme risklerine dikkat çekmektedir., This study was conducted to determine the presence of fungi and water molds in 13 state and 3 private nurseries in Sakarya, Kocaeli, Yalova, Bursa, Istanbul, Kırklareli and Edirne provinces where woody plants were produced. For this purpose, the coniferous and deciduous seedlings showing various disease symptoms, especially death and drying, were sampled together with their soil. Laboratory studies included microscopic investigations and isolation of fungi from diseased tissues and isolation of Pythiaceous species from soil samples of sampled seedlings. Two hundred twenty-seven deciduous and coniferous seedlings and 79 soil samples were taken from the nurseries. Totally 145 fungal and 57 Pythiaceous isolates were obtained. The isolates were grouped into morphotypes based on their morphological characteristics, and representative isolates were identified by ITS-based DNA analysis. Marssonina brunnea, Melampsora allii-populina on poplar and Dothistroma septosporum on pine were the fungal disease agents detected based on their morphological and microscopical properties. In addition to these species, Fusarium solani, Fusarium oxysporum, Pythium aphanidermatum, Pythium irregulare, Pythium ultimum, Phytopythium vexans and Phytophthora syringae were among other important and common species identified. The surveys and laboratory studies conducted within the scope of this research showed that state nurseries host more fungal and Pythiaceous species than private nurseries. The amount and diversity of the obtained fungal and oomycete species draws attention to the risk that these microorganisms can be transported from one nursery to another and even to afforestation areas.

An investigation study was carried to test the ability of endophytic bacteria isolated from small millets as a biocontrol agent against the fungal pathogen Pythium aphanidermatum, the causal organism of sheath blight in foxtail millet (Setaria italica L.) grown in seedling trays under greenhouse conditions. In total twelve bacterial endophytes were isolated out of which six isolates produced β -1, 3 glucanases, 11 isolates produced chitinases and 12 isolates produced siderophores under in-vitro conditions. All these bacterial endophytic isolates inhibited mycelial growth of Pythium aphanidermatum in plate assay and liquid culture and the highest percent inhibition of mycelium was recorded in KMS5 (44.44%) followed by KMS1 (38.89%). In seedling trays, BMR7 and KMS5 (81.66%) had recorded the highest percent germination followed by KMS1 (79.62%) and KMS1 and KMS5 took 10.85 and 10.55 days respectively for 50% seed germination compared to control (13.50 days) which was treated with pathogen alone. Lowest pre-emergence disease incidence was noticed in KMS5 (14.03%) followed by KMS1 (16.18%) whereas T1 (Control) recorded maximum pre-emergence disease incidence (39.82%) was observed. Least post-emergence disease incidence (11.36) and biocontrol efficacy (68.74%) was recorded with KMS5. Apart from showing antagonistic activity, KMS5 had recorded a significantly higher seedling vigor index (2712.97), root length (18.12 cm), shoot length (15.10 cm), root dry weight (0.158 g) and shoot dry weight (0.76 g) compared to other bacterial endophytic isolates.

The efficacy of bacterial isolates obtained from the rhizosphere soil samples collected from cucumber fields of Muscat Governorate of Oman was assessed against Pythium aphanidermatum, the causal agent of damping-off of cucumber. Of the 58 isolates screened in a dual culture assay, only four isolates (B11, B9, AT3 and 4A) inhibited the growth of P. aphanidermatum and produced inhibition zones of above 5 mm. Among the four isolates, B11 was the most effective followed by B9, AT3 and 4A, which recorded inhibition zones of 14 mm, 13 mm, 11 mm and 6.8 mm, respectively. Scanning electron micrographs of hyphae of P. aphanidermatum grown in the presence of antagonists showed morphological abnormalities. These antagonistic bacteria were identified based on sequence analysis of 16S rDNA gene. The potential of the promising antagonists, Pseudomonas resinovorans (B11) and Pseudomonas aeruginosa (AT3) was evaluated either individually or in combination with a commercial formulation of Trichoderma viride against damping-off of cucumber under greenhouse conditions. Among the biocontrol treatments, soil application of P. resinovorans (B11) was the most effective, which resulted in a significantly lower damping-off incidence (24 to 30%) compared with Pythium-infected control (82 to 84%). To our knowledge, this is the first report on biological control of P. aphanidermatum with P. resinovorans.

Using antagonistic bacterium is an effective method to control plant disease by fungal pathogens. An aerobic bacterium designated SJ-25, capable of suppressing Fusarium graminearum, Exserohilum turcicum, Pythium aphanidermatum, and Cochliobolus sativus, was isolated from farmland soil. The phylogenetic analysis revealed that strain SJ-25 belongs to the species of Sphingobacterium psychroaquaticum. The genome of strain SJ-25 consists of a 4,396,535-bp chromosome with a G+C content of 41.7 mol%; including 3696 CDS, 64 tRNA genes and six rRNA operons. Genomic analysis revealed that its genome contains multiple genes responsible for biosynthesis of siderophore, methyl 4-hydroxybenzoate, chitinase, giving strain SJ-25 the antagonistic ability on fungi pathogen. Strain SJ-25 harbors sets genes responsible for production of 2, 3-butanediol and salicylic acid, which could elicit the induced systemic resistance of the host plant. This genome sequence could be used as a basis material for further exploration of antagonistic mechanisms on fungi, widening our understanding of the ecological role of the genus Sphingobacterium in farmland ecosystem.

A total of 24 morphologically distinct salt-tolerant bacteria were isolated from the tomato rhizosphere soil and tested in vitro against Pythium aphanidermatum, causing damping-off disease in tomat...

To develop natural fungicides against Panax notoginseng disease, the antifungal effects of Syzygium aromaticum essential oils (EOs) against the pathogenic fungi causing P. notoginseng disease were investigated. The effects of EOs from S. aromaticum buds and fruits on the growth of pathogenic mycelia were determined with in vitro antifungal experiments using the Oxford cup method. The results suggested that S. aromaticum EOs had a strong inhibitory effect on the growth of pathogenic fungi. At a dose of 50 mg/mL, the inhibitory effects of S. aromaticum buds EOs was greater than those of S. aromaticum fruits. Then the chemical constituents of the S. aromaticum EOs were analyzed by GC–MS. The results indicated that the eugenol content was the highest, accounting for 57.95% of the total constituents in the S. aromaticum buds EOs and 39.26% in the S. aromaticum fruits EOs. The minimum inhibitory concentration (MIC) of EOs, the major constituents, and hymexazol were evaluated. In addition, the antifungal effect of their combination was also determined by the fractional inhibitory concentration (FIC). When the buds EOs were combined with hymexazol, they had a synergistic effect in inhibiting Pythium aphanidermatum, Botrytis cinerea, and Colletotrichum gloeosporioides. The combination of S. aromaticum fruit EOs with hymexazol showed an additive effect on Fusarium solani, P. aphanidermatum, and B. cinerea. The preventive effect of S. aromaticum bud EOs combined with hymexazol was up to 60.77% in in vivo experiments. Overall, S. aromaticum EOs had a broad spectrum of antifungal activities against pathogenic fungi influencing the growth of P. notoginseng, which would provide a scientific basis for the ecological control of P. notoginseng disease.

Pythium spp. are the causal agents of Pythium root rot and damping-off on microgreens. The objective of this project was to assess the efficacy of biofungicides on Pythium root rot and damping-off caused by Pythium aphanidermatum and Pythium dissotocum on microgreens in greenhouses. In the first experiment, arugula (Eruca sativa Mill.), kale (Brassica oleracea var. sabellica L.), radish (Raphanus raphanistrum subsp. sativus L.), and mustard (Brassica juncea L. Czern) microgreens were treated with Companion® (Bacillus subtilis GB03), Triathlon BA® (Bacillus amyloliquefaciens D747), or RootShield Plus® (Trichoderma harzianum KRL-AG2 and Trichoderma virens G-41) in a hydroponic nutrient film technique system. Two days after treatment, the plants were inoculated with 3 × 105 zoospores of Pythium spp. After seven days, we measured root necrosis, damping-off incidence and severity, and plant biomass. All plants infected with Pythium spp. were smaller by 28% or more compared with non-inoculated plants. Overall disease was low, but biomass was lower in all treatments inoculated with Pythium spp. Arugula infected with Pythium spp. and treated with Triathlon BA® resulted in 8% lower disease incidence compared with the positive control, yet Triathlon BA® resulted in the highest root necrosis. On a separate experiment, arugula and mustard were grown in propagation trays, irrigated manually, and treated with the biofungicides mentioned above or Cease® (Bacillus subtilis QST 713). Arugula and mustard plants inoculated with Pythium spp. had 74.4% reduction of shoot dry weight. Arugula and mustard treated with Cease®, with and without Pythium spp., resulted in ≥59% more biomass compared with the untreated inoculated control. In the tray experiment, all the infected plants treated with biofungicides had more biomass than plants with no biofungicides. Results from this experiment suggest that microbial biofungicides can be introduced in nutrient solutions in nutrient film technique or applied in the irrigation to prevent Pythium root rot and damping-off in brassica microgreens. However, biofungicides can reduce plant biomass and growers may need to extend production time to achieve target yields.

Occurrence and cucumber damping-off disease incidence was determined in Sulaimani plastic houses in 2014 revealed from overall disease incidence of 6.82%. The highest incidence and severity reached to 23.7% and 5.0 respectively in Kharajian. While the lowest incidence and severity was detected in Arabit (0.2% and 0.6 respectively). Disease symptoms include pre and post-emergency damping-off of cucumber seedlings. Twelve fungal pathogens were isolated from roots and crown of infected seedlings and plants that explore typical damping-off and root rot symptoms. Rhizoctonia solani was the most frequently isolated fungi followed by Pythium aphanidermatum, Fusarium solani and Pythium sp. Morphology and characteristics of R. solani and P. aphanidermatum match with the original described characters of the fungi. The optimum growth temperature for P. aphanidermatum was 30°C and for R. solani was between 25-30°C. Pathogenicity test revealed that R. solani significantly surpassed all other treatment except P. aphanidermatum by inciting 53.3% pre and 66.4% post-emergency damping-off followed by P. aphanidermatum that incited 43.6% and 56.3% pre and post emergency damping-off respectively. T. harzianum showed high antagonistic ability against both pathogens. Antagonistic ability degree of T. harzianum reached to 37.02 against P. aphanidermatum and 32.00 against R. solani. The bio-control bacterial Bacillus subtilis, Rhizobacteria, Streptomyces coelicolor showed high efficiency in controlling the disease. Rhizobacteria and S. coelicolor completely inhibit R. solani growth at 10-1 bacteria dilution and significantly surpassed all other treatments. dilution 10-1 from all the used bacteria were significantly more efficient against P. aphanidermatum. This dilution was contain 21.4 × 107 cell forming unit in each milliliter (CFU/ml) in B. subtilis, 28 × 107(CFU/ml) in Rhizobacteria, 29.5 × 107 (CFU/ml) in S. coelicolor, 32.2× 107(CFU/ml) in Pseudomonas flouresence and 22.6 × 107 (CFU/ml) in Azotobacter chroococcus.

Considering the different carbonic source utilisation abilities of microbial species, we used six carbonic supplements to acclimate the microorganisms in two types of compost teas (named CT...

The aim of this study was to develop a multiplex PCR assay for rapid, reliable and simultaneous detection of five important cucumber pathogens: Fusarium oxysporum, F. solani, Sclerotinia sclerotiorum, Pythium aphanidermatum and Botrytis cinerea. One common forward primer and species-specific reverse primers were designed based on comparative genomics. The specificity of the developed multiplex PCR was confirmed using target pathogens and other closely related pathogens. Furthermore, this method accurately identified the presence of pathogens among cucumber field samples. Consequently, we established a multiplex PCR method which could provide reliable and informative identification of five cucumber pathogens in the tested environmental samples.

Pythium oligandrum is one of the best known fungi in biological control of many fungal plant diseases. The objective of this study was conducted to highlight the possibility of using cultural filtrate of this fungus in inhibiting the growth of reproducting units of the pathogenic Pythium aphanidermatum and P. diclinum. Two isolates of each fungus were studied. Ability of P. oligandrum to grow and develop on V-8 liquid medium was tested in order to study its effect on mycelium growth, zoospore and oospore production of each of the pathogenic pythia of P. aphanidermatum and P. diclinum. Cultural filtrate of P. oligandrum (quarter and half concentration) significantly inhibited mycelial growth and the production of zoospores and oospores of the pathogenic tested fungi. The rate of inhibition in all treatments reached more than 90% in case of the use of half concentration of the cultural filtrate of P. oligandrum. The study will provide information for the use of cultural filtrate of potential selected isolates of P. oligandrum in biological control of some fungal diseases especially in aquaculture. The outcomes of this research can be easily applied in aquatic farming but need further work on different types of pathogenic fungi.

Bacillus sp is one of the biocontrol agents are extensively used in management of fungal diseases of crop plants, exhibiting mycoparasitism against a wide range of plant pathogens. In the present investigation Efficacy of Bacillus subtilis was tested against Pythium aphanidermatum under glass house and field conditions. With regard to the germination and seedling growth parameters, the treatment T2 (liquid formulation of Bacillus subtilis @10 ml/L) recorded the highest germination percentage (92.59%), shoot length (59.3 cm), root length (24.3 cm), vigor index (7740.52) and yield (22.67 mt/ha). This was followed by the treatments T3, T1 and T4 in the decreasing order of merit. In the biometric observations also, 10 ml/L and 20 ml/L concentration of liquid formulation of Bacillus subtilis recorded statistically significant results. Observations on total protein content in brinjal plants treated with different concentrations of Bacillus subtilis revealed that treatment T2 was found best in both field and glass house conditions yielded highest protein as 0.44mg/ml and 0.30mg/ml respectively. The least values of germination, growth parameters, protein content and yield were observed in untreated control.

Tomato (Solanum lycopersicum L.), one of the most widely grown vegetables worldwide, is susceptible to damping-off and root rot caused by Pythium aphanidermatum (Edson) Fitzpatrick. In in vitro assays, five Trichoderma isolates, viz., Trichoderma harzianum (Th), T. asperellum (Ta), T. virens (Tvs1), T. virens (Tvs2) and T. virens (Tvs3) were compared for their ability to suppress P. aphanidermatum. The mycelial growth of the pathogen was inhibited in vitro after placing each Trichoderma species and isolates on the opposite sides of the same Petri dish. Trichoderma isolates were able not only to arrest the spread of the pathogen but also invade the surface of its colony and sporulate over the colony. Additionally, conidia of Trichoderma isolates were able to inhibit the germination of zoospores of P. aphanidermatum in vitro. Control of tomato damping-off and root rot diseases by soil treatment with the inoculum preparations of Trichoderma isolates employed either alone or in combination was attempted. In greenhouse experiment, the combined inoculation of five Trichoderma isolates suppressed damping-off induced by P. aphanidermatum and increased the survival of tomato plants by 74.5%. In field experiment, the possibility of reducing plant death resulting from root rot disease caused by P. aphanidermatum using Trichoderma isolates, employed either alone or in combination, was investigated. The combined inoculation of five Trichoderma isolates was the most effective treatment, decreasing root rot by 57.2% and increasing the survival of tomato plants by 87.5%. The tested Trichoderma isolates stimulated systemic defence responses in tomato plants grown in the field by activating defence enzymes including peroxidase, polyphenoloxidase and chitinase. Additionally, the chlorophyll contents in the leaves of treated tomato plants were markedly increased. Moreover, the combined inoculation of the five isolates yielded the highest records of growth parameters and fruit yield compared with individual inoculation. Therefore, it was concluded that the mixture containing Trichoderma species and isolates may be used to control damping-off and root rot of tomato caused by Pythium aphanidermatum.

Our purpose was to evaluate the ability of Trichoderma aggressivum f. europaeum as a biological control agent against diseases from fungal phytopathogens. Twelve isolates of T. aggressivum f. europaeum were obtained from several substrates used for Agaricus bisporus cultivation from farms in Castilla-La Mancha (Spain). Growth rates of the 12 isolates were determined, and their antagonistic activity was analysed in vitro against Botrytis cinerea, Sclerotinia sclerotiorum, Fusarium solani f. cucurbitae, Pythium aphanidermatum, Rhizoctonia solani, and Mycosphaerella melonis, and all isolates had high growth rates. T. aggressivum f. europaeum showed high antagonistic activity for different phytopathogens, greater than 80%, except for P. aphanidermatum at approximately 65%. The most effective isolate, T. aggressivum f. europaeum TAET1, inhibited B. cinerea, S. sclerotiorum, and M. melonis growth by 100% in detached leaves assay and inhibited germination of S. sclerotiorum sclerotia. Disease incidence and severity in plant assays for pathosystems ranged from 22% for F. solani to 80% for M. melonis. This isolate reduced the incidence of Podosphaera xanthii in zucchini leaves by 66.78%. The high compatibility by this isolate with fungicides could allow its use in combination with different pest management strategies. Based on the results, T. aggressivum f. europaeum TAET1 should be considered for studies in commercial greenhouses as a biological control agent.

SummaryPseudomonas aeruginosa is a leading opportunistic pathogen capable of causing fatal infections in immunocompromised individuals and patients with degenerative lung diseases. Agricultural soil and plants are the vast reservoirs of this dreaded pathogen. However, there have been limited attempts to analyze the pathogenicity of P. aeruginosa strains associated with edible vegetable plants. This study aims to elucidate the virulence attributes of P. aeruginosa strains isolated from the rhizosphere and endophytic niches of cucumber, tomato, eggplant, and chili collected from agricultural fields. Virulence of the agricultural strains was compared to three previously characterized clinical isolates. Our results showed that 50% of the plant-associated strains formed significant levels of biofilm and exhibited swarming motility. Nearly 80% of these strains produced considerable levels of rhamnolipid and exhibited at least one type of lytic activity (hemolysis, proteolysis, and lipolysis). Their virulence was also assessed based on their ability to suppress the growth of plant pathogens (Xanthomonas oryzae, Pythium aphanidermatum, Rhizoctonia solani, and Fusarium oxysporum) and kill a select nematode (Caenorhabditis elegans). The plant-associated strains showed significantly higher virulence against the bacterial phytopathogen whereas the clinical strains had significantly higher antagonism against the fungal pathogens. In C. elegans slow-killing assay, the clinical strains caused 50-100% death while a maximum of 40% mortality was induced by the agricultural strains. This study demonstrates that some of the P. aeruginosa strains associated with edible plants harbor multiple virulence traits. Upon infection of humans or animals, these strains may evolve to be more pathogenic and pose a significant health hazard.