Pythium aphanidermatum causing seedling damping-off of Hylocereus megalanthus in China.

Hylocereus megalanthus (family Cactaceae), commonly known as bird's nest fruit (Yanwo fruit), was a new tropical plant cultivated commercially in south China because of its high nutritional content and sweet taste. In August 2023, damping-off disease of approximately 60% of seedlings was observed at a nursery in Zhanjiang, Guangdong Province (E110°17'46″ N21°9'2″). Stems of infected seedlings exhibited symptoms of water-soaked tissue which caused collapse at the base of the stem and sloughing of necrotic root cortex tissue was observed (Figure 1). White aerial mycelia were visible on the surface of the stem and soil at a high relative humidity. Diseased tissues about 0.5 cm2 were taken from the infected roots and stems, surface disinfected with 75% ethanol and 3% hydrogen peroxide solution, each for 1 min, subsequently rinsed in sterile water, and placed on potato dextrose agar (PDA). Plates were incubated at 25 to 28℃ in the dark for 3 days. Coenocytic hyphae grew from all infected roots and stems. Hyphal tip transfers were completed twice, and twelve isolates with the same morphological characteristics were obtained. The colony growth on PDA was ample. Main hyphae are up to 9.5 µm wide. Sporangia were terminal, inflated, branched or unbranched. Encysted zoospores were 7.5 µm in diameter. Oogonia were terminal, globose, smooth and of 16.8 to 27.4 µm (average 21.5 µm) diameter. Oospores were typically spherical, thick-walled, yellowish, 19.7 to 26.3 µm (average 21.1 µm) diameter, wall 1 to 2 µm thick. Antheridia were mostly intercalary, sometimes terminal, broadly sac-shaped, 15.0×19.0 µm (Figure 2). The morphological features were very similar to those of Pythium spp. (Toporek and Keinath 2021). For further identification, the LSU and ITS regions of isolate CCAS-YWGCD (stored in Agricultural Culture Collection of China, ACCC 35633) were amplified and sequenced with using primer pairs LROR/LR7 and ITS1/ITS4, respectively (Gao et al. 2017; White et al. 1990). The resulting sequences were deposited in GenBank (ITS: OR775664; LSU: OR775667). BLASTn results showed 100% sequence similarity with reference sequences of Pythium aphanidermatum (AY598622 for ITS and HQ665084 for LSU). Phylogenetic tree generated from maximum likelihood analysis based on combined LSU and ITS sequence data with MEGA 10.1.8, clustered the oomycete in P. aphanidermatum clade with 100% bootstrap support (Figure 3). Therefore, the oomycete was identified as P. aphanidermatum. To confirm Koch's postulates, six three-month-old seedlings of H. megalanthus (height about 15 cm) were transplanted to 15 cm pots. Six-mm-diameter mycelial plugs obtained from 7-day-old cultures at 25℃ in the dark were buried adjacent to the stem of three unwounded healthy seedlings. Another three seedlings inoculated with PDA agar served as controls. The plants were covered with plastic bags, kept at about 30℃, and watered regularly to keep the soil moisture content high. All inoculated seedlings exhibited symptoms of stems rot and damping-off, Symptoms did not develop on the control seedlings. P. aphanidermatum by morphological and molecular analysis was reisolated from the stems. P. aphanidermatum had been reported worldwide causing disease in many agricultural crops (Qi et al. 2021; Kim et al. 2020), but this is the first report causing damping-off of H. megalanthus seedling in China as well as worldwide, and this disease should be monitored in nursery seedlings.

Development of biocomposite films incorporated with the extract from pitcher associated bacteria for the postharvest protection from fungi.

Pythium aphanidermatum is known to cause diseases like damping-off, root rot, stem rot and fruit rot in a wide range of plants. Eventhough many chemical methods have been demonstrated to have the potential to manage these diseases, their benefits are being offset equally by the negative side effects. Therefore, the control of Pythium spp. using natural antifungal agents is of immense significance due to its environmental safety. Here, the plant associated microorganisms with antifungal metabolites have significant promises to be explored both as sustainable biocontrol agents and also as active constituents of antifungal materials. Antimicrobial packaging films prepared using such components can have significant applications to meet the requirements to prevent postharvest loss of agricultural produce by inhibiting the fungal growth. Eventhough there are reports on the development of antimicrobial packaging films for such applications, the use of bacterial extracts with antifungal activity for the same is least investigated. Hence, the present study demonstrates the development of biocomposite films prepared using polyvinyl alcohol (PVA) incorporated with the extracts prepared from bacterial isolates (Serratia sp. NhPB1, Kocuria sp. NhPB49, and Pantoea dispersa NhPB54) previously isolated from the pitcher plant. Here, the individual films were prepared by incorporating 1 mL of bacterial extract in 40 mL of 3% PVA solution and the developed films were then subjected to antifungal activity screening against P. aphanidermatum. The antifungal activity analysis of the films prepared with the incorporation of extracts from Serratia sp. NhPB1, Kocuria sp. NhPB49, and Pantoea dispersa NhPB54 showed remarkable activity against the tested pathogen. The application of biocomposite films on Solanum lycopersicum and Capsicum annuum fruits for its protection from P. aphanidermatum by dip coating method further indicates the promises of developed biocomposite films for active packaging applications.

Occurrence of root rot caused by Pythium aphanidermatum on mung bean (Vigna radiata) in China.

In Aug 2019, approximately 10% of mung bean plants at the experimental farm of the Jiangsu Academy of Agricultural Science (32.03 N; 118.88 E) showed symptoms of stunting and wilting. Brown and water-soaked stem lesions were often observed at the base of the diseased plants. In severe cases, the plants collapsed and cumulous aerial mycelia were visible on the basal stem surface (Figure S1 A). To identify the causal agent, a total of 20 tissue fragments (5 mm long) were excised from roots and basal stems of five symptomatic plants. The fragments were surface sterilized in 2% sodium hypochlorite solution then plated on 2.5% potato dextrose agar (PDA) plates containing 10 µg/mL pimaricin, 100 µg/mL ampicillin, 10 µg/mL rifampicin, and 10 µg/mL pentachloronitrobenzene (PARP; Beckerman et al. 2017). After 3-4 days incubation at 25oC in dark, 14 colonies with white and cumulous mycelia grew from the tissue pieces (named as JS19-1 to JS19-14). JS19-1 and JS19-2 were purified by hyphal tipping, then grown on PDA medium for 7 days for morphological observation using a compound microscope (Figure S1 B, C). Width of coenocytic hyphae ranged from 3.7 to 8.9 (avg. 6.1, n=20) µm. Terminal oogonia were globose and with a diameter of 13.8 to 25.8 (avg. 22, n=20) µm. Antheridia were barrel-shaped, and mostly intercalary, sometimes terminal. Most of antheridia were diclinous, with 6.2 to 12.5 (avg. 9.3, n=20) µm in width and 7.6 to 15.3 (avg. 12.8, n=20) µm in length. Oogonia were fertilized with one or two (rare) antheridia. Oospores were aplerotic, 10.1 to 23.5 (avg. 20.4, n=20) µm in diameter. Sporangia had terminal inflated hyphal branches (Figure S1 D, E). The two isolates were preliminary identified as Pythium aphanidermatum. For molecular identification, the sequences of internal transcribed spacer (ITS) rDNA, cytochrome oxidase subunit I (CoxI) (Robideau et al. 2011), and ß-tubulin (Kroon et al. 2004) of JS19-1 were detected, and deposited in GenBank (MT949538, MT949539 and MT949540). The ITS and CoxI sequences were identical with P. aphanidermatum CBS28779 ITS (759/759 bp, HQ643439.1) and PYT01 CoxI (640/640 bp, MH760243.1) respectively, the ß-tubulin sequence showed 99% (830/840 bp) similarity of P. aphanidermatum P2 (AY564048.1). Thus, JS19-1 was confirmed as P. aphanidermatum. To fulfill Koch's postulates, the pathogenicity of JS19-1 was tested using the procedure of Kiyoshi et al. (2021) with some modifications. Barley grains infested with JS19-1 were as inoculum and thoroughly mixed with potting mixture at a rate of 10% in volume. Six mung bean seeds were sown per pot and then grown in a greenhouse. Potting mixture with no inoculum was used as control. Three pots of replicate plants used for inoculation and control. After 3 weeks, emergence in the inoculated pots was 33% and symptoms of stunting and root rot similar to those in field were observed, while control plants were asymptomatic (FigureS1 F, G). P. aphanidermatum was successfully reisolated from symptomatic plants of both methods. The pathogenicity tests were repeated twice. P. aphanidermatum causes seed rot, pre- and postemergence damping-off, or stem/root rot of a wide range of industrial crops and vegetables (Liu et al, 2018). To our knowledge, this is the first report of P. aphanidermatum causing disease on mung bean in China. Since Phytophthora vignae (Sun et al, 2020) and P. myriotylum (Yan et al, 2021) have been reported causing mung bean root rot, integrated disease management should be adopted to reduce damage.

First report of Pythium aphanidermatum causing root rot of head lettuce in China.

Head lettuce (Lactuca sativa L.) is an important crop for fresh consumption in China. In Shandong Province, head lettuce is planted in spring and in autumn each year. Because of the on-and-off rain for three weeks, head lettuce plants planted directly into the field in Jiyang City, in July 2017, 20% of the plants rapidly showed symptoms of rotting, water-soaked lesions on roots and stem bases, and then death. The diseased plants first appeared in low-lying areas prone to water accumulation. One-millimeter pieces were excised from water-soaked roots and stem bases, dipped in a 0.2% calcium hypochlorite solution for 10 min, then placed on V8 medium, and incubated in the dark at 28°C for 5 d. Two Pythium-like strains were isolated from the roots and stems. The isolates transferred to CMA and grown for 7 d, and the morphological characteristics of the two isolates on corn meal agar (CMA) were white with dense, cottony, aerial and well-branched mycelia. The two isolates produced sporangia, oogonia, antheridia and oospores. Most of the sporangia were lobate. The oogonia were smooth, nearly globose and terminal. Oospores were globose, smooth and aplerotic. The average dimensions of 50 oogonia and oospores respectively ranged from 19.5 to 25.2 (av. 23.1) µm and 17.8 to 22.3 (av. 19.9) µm. The antheridia were broadly sac-shaped. The isolates morphological characteristics were consistent with P. aphanidermatum (van der Plaats-Niterink, 1981). The COI gene and ITS region of the rDNA were amplified and sequenced using primers FM55/FM52R (Long et al. 2012) and ITS1/ITS4 (White et al. 1990), respectively. The two aligned COI sequences were identical for both isolates, as were the two ITS sequences. BLASTn analysis of the 1,133-bp COI sequence (accession no. MT952703) resulted in a 100% identity with accession number AY129164 from Lactuca sativa, which belongs to P. aphanidermatum, and the 808-bp ITS sequence (accession no. MT921597) showed a 99% identity with Genbank accession number HQ643442 belonging to P. aphanidermatum. Koch's postulates were conducted by first soaking corn kernels for 24 h in water, and then autoclaving for 2 h at 121˚C. Isolate SDHL-1 was grown on CMA for 10 days, after which agar plugs were transferred to the sterilized corn kernels and incubated at 28℃ for approximately 15 d, until the corn kernels were covered in white hyphae. Ten healthy head lettuce plants were transplanted into a sterilized loam potting soil artificially infested with the corn inoculum (3 g inoculum per 100 g loam mixture). Inoculated plants and noninoculated controls were maintained in a greenhouse at 28°C and 100% relative humidity with a 12-h photoperiod; the experiment was repeated once. All twenty inoculated plants exhibited symptoms within one week similar to those observed. Pythium aphanidermatum was recovered only from the water-soaked roots and stem bases of inoculated plants and the re-isolated cultures again identified based on morphological characteristics and sequencing of the ITS and COI genes. No symptoms were observed on the control plants. Sclerotinia sclerotiorum is reported to cause stem base rot of L. sativa in China (Zhou et al. 2011). To our knowledge, however, this is the first report of root rot of head lettuce caused by Pythium aphanidermatum. Identification of the pathogen will assist in devising strategies to reduce yield loss.

First Report of Damping-off of Ovate-leaf Atractylodes Caused by Pythium aphanidermatum in South Korea.

Ovate-leaf Atractylodes (Atractylodes ovata) is a well-known medicinal plant in Korea, where the dried rhizome and the root extract are used in herbal medicines. In 2019, severe damping-off of ovate-leaf Atractylodes at the early seedling stage was observed in a commercial planting in Sangju, South Korea. Approximately 35% of young seedlings suddenly wilted and then died despite adequate soil moisture. Putative causal agents were isolated from roots of diseased plants. Roots were washed thoroughly wih tap water, cut into 1-cm-long pieces and then split in half longitudinally. Root pieces were surface disinfected in 0.1% NaOCl solution for 1 min, washed three times with sterilized distilled water, and finally blotted dry. Root tissue was placed on potato dextrose agar (PDA, Difco, and Becton Dickinson) amended with tetracycline (0.05 g/L) and incubated at 25°C in the dark. Twelve pure culture of the potential causal agent were obtained by hyphal tipping twice. Mycelia of the seven-day-cultures were white and the colony produced numerous oogonia. The oogonia were smooth, globose, terminal and rarely intercalary, and 17.3 to 27.1 µm (mean ± SD 24.3 ± 2.25 µm) in diameter. Antheridia were diclinous or monoclinous (rare) with clavate cells that were 17.9 to 31.6 × 7.1 to 8.8 µm (mean 26.8 × 7.5 µm). The stalks of the antheridia were unbranched. Oospores were plerotic or nearly plerotic and 16.3 to 25.4 µm (mean ± SD = 20.1 µm ± 3.2) in diameter. The morphological characteristics of the isolates were comparable to those of Pythium spp. including P. aphanidermatum (Van der Plaats-Niterink,1981). The internal transcribed spacers (ITS), cytochrome oxidase 1 (cox1), and cytochrome oxidase 2 (cox2) regions of genomic DNA from SRRS1, SRRS2, and SRRS4 were amplified using primer sets ITS5 + ITS4, FM52R + FM55, and FM58 + FM66 respectively (Ueta and Tojo 2016) and sequenced. Resulting sequences were deposited in GenBank under accession numbers LC569777 to LC569779 and LC569785 to LC569790. The concatenated sequence data contained 46 taxa for the ITS-cox2 phylogenetic analysis and 17 taxa for cox1. The maximum likelihood estimation and Bayesian inference consensus tree showed that the present isolates formed a clade with P. aphanidermatum (strain NBRC 100101, P36-3, and 1987-61). The pathogenicity of three isolates was tested using the procedure described by Herrero et al. (2003) with some modification. Five surface disinfected seeds were sown in a plastic pot containing autoclaved peat soil. There was a total of twelve pots. After emergence seedlings were thinned to three similar sized seedlings per pot and grown for 25 days at 25°C in the 16-h light. Seedling were then inoculated with mycelial plugs (10 mm diam.) from four day old cultures on PDA. Three plugs per pot were placed 1 cm apart from the seedlings (one plug per seedling). Three pots per isolates were inoculated and three pot with non-inoculated seedlings were used as control. All plants were grown in a growth chamber at 25°C with 16-h light and 80% relative humidity and irrigated twice per week with sterile water. All inoculated seedlings (100%) suddenly collapsed 10 days after inoculation while the control plants remained healthy and vigorous. Pythium aphanidermatum was re-isolated from the inoculated seedlings and identified following the procedures previously described thus completing Koch's postulates. This pathogen has also reported as the causal agent of damping-off of common bean, cucumber, wheat, tomato and tobacco (Al-Saadi et al. 2007; Herrero et al. 2003; and Gilardi et al. 2018). To our knowledge, this is the first report of Pythium aphanidermatum causing damping-off disease on ovate-leaf Atractylodes in South Korea, and this pathogen could threaten production. Outcomes of present study will help manage this disease with effective control measures.

Pythium aphanidermatum as a Novel Oomycete Pathogen Causing Leak on Potato (Solanum tuberosum) in Korea.

Potato (Solanum tuberosum L.) is one of the most important food crops in Korea. In July 2021 and 2022, dark black-rot symptoms with pink tinges were observed on field-grown potato tubers in Hongsung and Chuncheon, Korea, respectively. We obtained four isolates (HSv05 and HSv10 from Hongsung, and CCp03 and CCp05 from Chuncheon) from diseased tubers and identified these isolates as Pythium aphanidermatum by analyzing the sequences of internal transcribed spacer rDNA region and mitochondrial cytochrome c oxidase subunit II (COX2) mtDNA gene. Additionally, we compared the cultural and morphological characteristics of these four isolates with those of the reference isolate KACC 48066 of P. aphanidermatum and the literature. Further, we tested the pathogenicity of all these isolates against potato tubers. The cultural and morphological characteristics of the four test isolates were similar to those of the reference isolate and the literature; all four test isolates proved pathogenic to potato tubers. Therefore, we concluded that P. aphanidermatum is the causal agent of potato leak and this is the first report of the disease on potato in Korea.

Efficacy of Streptomyces murinus JKTJ-3 in Suppression of Pythium Damping-Off of Watermelon.

Damping-off caused by Pythium aphanidermatum (Pa) is one of the most destructive diseases for watermelon seedlings. Application of biological control agents against Pa has attracted the attention of many researchers for a long time. In this study, the actinomycetous isolate JKTJ-3 with strong and broad-spectrum antifungal activity was screened from 23 bacterial isolates. Based on the morphological, cultural, physiological, and biochemical characteristics as well as the feature of 16S rDNA sequence, isolate JKTJ-3 was identified as Streptomyces murinus. We investigated the biocontrol efficacy of isolate JKTJ-3 and its metabolites. The results revealed that seed and substrate treatments with JKTJ-3 cultures showed a significant inhibitory effect on watermelon damping-off disease. Seed treatment with the JKTJ-3 cultural filtrates (CF) displayed higher control efficacy compared to the fermentation cultures (FC). Treatment of the seeding substrate with the wheat grain cultures (WGC) of JKTJ-3 exhibited better control efficacy than that of the seeding substrate with the JKTJ-3 CF. Moreover, the JKTJ-3 WGC showed the preventive effect on suppression of the disease, and the efficacy increased with increase in the inoculation interval between the WGC and Pa. Production of the antifungal metabolite actinomycin D by isolate JKTJ-3 and cell-wall-degrading enzymes such as ß-1,3-glucanase and chitosanase were probably the mechanisms for effective control of watermelon damping-off. It was shown for the first time that S. murinus can produce anti-oomycete substances including chitinase and actinomycin D. This is the first report about S. murinus used as biocontrol agent against watermelon damping-off caused by Pa.

9-Tricosene Containing Blend of Volatiles Produced by Serratia sp. NhPB1 Isolated from the Pitcher Plant Provide Plant Protection Against Pythium aphanidermatum.

Plant-associated bacteria exhibit diverse chemical means to protect plants from the pathogens. The present study has been conducted to evaluate the volatile-mediated antifungal activity of Serratia sp. NhPB1 isolated from the pitcher plant against the notorious pathogen Pythium aphanidermatum. The study has also evaluated the protective effect of NhPB1 on Solanum lycopersicum and Capsicum annuum leaves and fruits against P. aphanidermatum. From the results, NhPB1 was found to have remarkable activity against the tested pathogen. The isolate was also found to impart disease protection in selected plants as evidenced by the morphological changes. Here, the leaves and fruits of S. lycopersicum and C. annuum control which were treated with the uninoculated LB and distilled water were found to have the presence of P. aphanidermatum growth with lesions and decaying of tissues. However, the NhPB1-treated plants did not show any symptoms of fungal infection. This could further be confirmed by the microscopical examination of tissues by propidium iodide staining. Here, the normal architecture of leaf and fruit tissues could be observed in the NhPB1-treated group, but the tissue invasion by P. aphanidermatum was observed in the control group which further confirms the promises of selected bacteria for biocontrol applications.

Bioactive Metabolites of Serratia sp. NhPB1 Isolated from Pitcher of Nepenthes and its Application to Control Pythium aphanidermatum.

Plant-associated bacteria have already been considered as the store house of bioactive compounds that confer the plant growth promotion and disease protection. Hence, the unique plant parts have already been expected to harbor diverse microbial communities with multi-beneficial properties. Based on this, the current study has been designed to identify the potential of Serratia sp. NhPB1 isolated from the pitcher of Nepenthes plant for its activity against the infamous pathogen Pythium aphanidermatum. The in vitro antifungal, plant growth promoting and enzymatic activities of the isolate indicated its promises for agricultural application. The isolate NhPB1 was also demonstrated to have positive effect on Solanum lycopersicum and Capsicum annuum, due to its plant beneficial metabolites. From the results of LC-MS/MS analysis, the isolate has also been revealed to have the ability to synthesize bioactive compounds including salicylic acid, cyclodipeptides, acyl homoserine lactone, indole-3-acetic acid, and serrawettin W1. These identified compounds and their known biological properties make the isolate characterized in the study to have significant promises as an eco-friendly solution for the improvement of agricultural productivity.

Plant-Disease-Suppressive and Growth-Promoting Activities of Endophytic and Rhizobacterial Isolates Associated with Citrullus colocynthis.

This study was conducted to investigate the antagonistic potential of endophytic and rhizospheric bacterial isolates obtained from Citrullus colocynthis in suppressing Fusarium solani and Pythium aphanidermatum and promoting the growth of cucumber. Molecular identification of bacterial strains associated with C. colocynthis confirmed that these strains belong to the Achromobacter, Pantoea, Pseudomonas, Rhizobium, Sphingobacterium, Bacillus, Sinorhizobium, Staphylococcus, Cupriavidus, and Exiguobacterium genera. A dual culture assay showed that nine of the bacterial strains exhibited antifungal activity, four of which were effective against both pathogens. Strains B27 (Pantoea dispersa) and B28 (Exiguobacterium indicum) caused the highest percentage of inhibition towards F. solani (48.5% and 48.1%, respectively). P. aphanidermatum growth was impeded by the B21 (Bacillus cereus, 44.7%) and B28 (Exiguobacterium indicum, 51.1%) strains. Scanning electron microscopy showed that the strains caused abnormality in phytopathogens' mycelia. All of the selected bacterial strains showed good IAA production (>500 ppm). A paper towel experiment demonstrated that these strains improved the seed germination, root/shoot growth, and vigor index of cucumber seedlings. Our findings suggest that the bacterial strains from C. colocynthis are suppressive to F. solani and P. aphanidermatum and can promote cucumber growth. This appears to be the first study to report the efficacy of these bacterial strains from C. colocynthis against F. solani and P. aphanidermatum.

Plant Protection Mediated Through an Array of Metabolites Produced by Pantoea dispersa Isolated from Pitcher Plant.

In the study, the bacterial isolate NhPB54 purified from the pitcher of Nepenthes plant was observed to have activity against Pythium aphanidermatum by dual culture and well diffusion. Hence, it was subjected to 16S rDNA sequencing and BLAST analysis, where the NhPB54 was found to have 100% identity to Pantoea dispersa. Upon screening for the plant beneficial properties, Pantoea dispersa NhPB54 was found to be positive for phosphate, potassium and zinc solubilization, nitrogen fixation, indole-3-acetic acid, ammonia, 1-aminocyclopropane-1-carboxylate deaminase, biofilm and biosurfactant production. Further to this, Solanum lycopersicum seedlings primed with P. dispersa NhPB54 were studied for the improved plant growth and disease protection. Here, the seedlings pre-treated with the NhPB54 culture supernatant were found to have enhanced plant growth and protection from damping off and fruit rot caused by P. aphanidermatum. From the LC-QTOF-MS/MS and GC MS analysis, P. dispersa NhPB54 was found to produce a blend of chemicals including 1-hydroxyphenazine, surfactin, and other bioactive metabolites with the likely basis of its observed antifungal and plant growth-promoting properties. From the results of the study, plants with unique adaptations can expect to harbor microbial candidates with beneficial applications.

Experimental Pythium aphanidermatum Infection in Rabbits.

Pythium aphanidermatum is a common plant pathogen, reportedly capable of human infection. This study investigated whether P. aphanidermatum can infect domestic rabbits. Four rabbits were inoculated with P. aphanidermatum subcutaneously and evaluated for 30 days. Blood and tissue samples were collected from all animals. The animals developed palpable nodules 5-10 days after inoculation. Necropsy revealed caseous nodular lesions characterized histopathologically by pyogranulomatous inflammation containing hyphae in the necrotic areas. P. aphanidermatum was isolated from the caseous material and anti-P. aphanidermatum antibodies were detected by serological examination. This is the first report of experimental P. aphanidermatum infection in mammals. The results demonstrate that this oomycete can produce infection at the inoculation site and induce an immune response. The results suggest that P. aphanidermatum may be undergoing adaptation to vertebrate hosts.

Activity of Clove Oil and Chitosan Nanoparticles Incorporated PVA Nanocomposite Against Pythium aphanidermatum.

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.

Mycogenic Nano-Complex for Plant Growth Promotion and Bio-Control of Pythium aphanidermatum.

(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.

Vascular pythiosis caused by Pythium aphanidermatum: the first case report in Asia.

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.

Effect of zinc oxide nanoparticle supplementation on the enhanced production of surfactin and iturin lipopeptides of endophytic Bacillus sp. Fcl1 and its ameliorated antifungal activity.

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.

Impact of Supplements on Enhanced Activity of Bacillus amyloliquefaciens BmB1 Against Pythium aphanidermatum Through Lipopeptide Modulation.

The present study has been designed to improve the activity of endophytic Bacillus amyloliquefaciens BmB1 against Pythium aphanidermatum through the culture supplementation with carbon sources, nitrogen sources and zinc oxide nanoparticles (ZnONPs). From the results of the study, supplementation with glucose (45 g/L), yeast extract (7.5 g/L) and ZnONPs (5 mg/L) were found to enhance the antifungal activity of B. amyloliquefaciens BmB1. This was also confirmed by comparative statistical analysis with experimental control. Further LC-Q-TOF-MS analysis of extracts of B. amyloliquefaciens BmB1 cultured with supplements showed a remarkable modulation of its lipopeptide profile. The blend of lipopeptides enhanced during the culture supplementation of B. amyloliquefaciens BmB1 as evidenced by the mass spectrometric analysis can consider to be the basis of its increased activity against P. aphanidermatum. As Bacillus spp. are well known for their biocontrol activities, the results of the study offer ways to improve its agricultural applications.

Synthesis and application of chitosan-copper nanoparticles on damping off causing plant pathogenic fungi.

Damping-off disease in seeds and young seedlings in agricultural crops is a major fungal disease that limits the agriculture production. Frequent use of synthetic fungicides against damping-off diseases is known to hamper the environmental balance. Thus, an alternative approach needs to be explored for the management of such economically important fungal diseases. In the present study, simple, economically feasible chitosan-coupled copper nanoparticles (Ch-CuNPs) were synthesized and demonstrated antifungal activity against damping-off disease causing phytopathogens, Rhizoctonia solani and Pythium aphanidermatum. Physico-chemical studies confirmed the size, shape, surface charge, element confirmation and mono-dispersed nature of Ch-CuNPs. In vitro efficacy studies revealed up to 98% mycelial growth inhibition at 0.1% Ch-CuNPs. An extracellular conductivity study of the mycelium showed cellular content leakage within 12 h of treatment. Further, plant toxicity study against chili, cowpea and tomato plants; showed that ≤0.2% NPs were safe under greenhouse conditions. NPs also exhibited growth-promoting activity with chili seeds, by overcoming the limited germination rate of susceptible seeds. Overall, the present study emphasizes the benefits of synthesized Ch-CuNPs on agricultural crops as fungicide and growth-promoter, as well as a safe alternative to pesticides in order to avoid hazardous effect on the environment.

Microbial Origin of Aquaponic Water Suppressiveness against Pythium aphanidermatum Lettuce Root Rot Disease.

Aquaponic systems are an integrated way to produce fish and plants together with mutual benefits. Fish provide nutrients to plants on the one side, and plant nutrients uptake allow water reuse for fish on the other side. In this kind of system, the use of phytosanitary treatments to control plant pathogens is sensitive because of the risk of toxicity for fish present in the same water loop, especially coupled aquaponics. Among plant pathogens, Pythium aphanidermatum is a most problematic microorganism due to the Oomycete's capacity to produce mobile form of dispersion (zoospores) in the recirculated water. Therefore, this study aimed at elucidating the potential antagonistic capacity of aquaponic water against P. aphanidermatum diseases. It was shown that aquaponic water presented an inhibitory effect on P. aphanidermatum mycelial growth in in vitro conditions. The same result was observed when lettuce plants growing in aquaponic water were inoculated by the same plant pathogen. Aquaponic lettuce was then compared to lettuce grown in hydroponic water or complemented aquaponic water (aquaponic water plus mineral nutrients). The disease was suppressed in the presence of aquaponic water, contrary to lettuce grown in hydroponic water or complemented aquaponic water. Root microbiota were analyzed by 16S rDNA and ITS Illumina sequencing to determine the cause of this aquaponic suppressive action. It was determined that the diversity and the composition of the root microbiota were significantly correlated with the suppressive effect of aquaponic water. Several taxa identified by metabarcoding were suspected to be involved in this effect. Moreover, few of these microorganisms, at the genus level, are known to have an antagonistic effect against P. aphanidermatum. These innovative results indicate that aquaponic water could be an interesting and novel source of antagonistic agents adapted to control P. aphanidermatum diseases in soilless culture.