Root rot of spinach caused by Pythium myriotylum and P. aphanidermatum in Taiwan.

Spinach (Spinacia oleracea) is a commonly used green vegetable. During September and October in both 2022 and 2023, a vegetable nursery company located among paddy rice fields in Taichung City, Taiwan, reported significant failures in spinach seedling production in net-houses with mean outdoor temperatures of 28.7℃. Abnormal growth was observed in approximately 30% of the spinach seedlings in each batch (n = 2,000 to 3,000), with aboveground tissues showing stunting, yellowing, and wilt, and underground tissues displaying root rot. The symptoms resembled the spinach damping-off documented in Taiwan in extension articles but which lacked complete pathogen identification. A total of 110 plants from two batches were used for pathogen isolation by placing roots on water agar incubated at 25℃ or were examined for the presence of oospores in diseased roots. Eighty-one percent of these plants were associated with Pythium. Nine Pythium isolates were used in subsequent analyses. Genomic DNA from these isolates was subjected to amplification of ITS, ß-tubulin gene (TUB2), and cytochrome C oxidase subunit Ⅱ (COXII) gene with primer pairs ITS1 / ITS4, BT5 / BT6, and FM58 / FM66 (Villa et al. 2006). Sequences of ITS (PP209187-PP209195), TUB2 (PP212864-PP212872), and COXII (PP212855-PP212863) were deposited in GenBank. Four isolates (sp01, sp02, sp03, and sp04) were 100% identical to the neotype strain (CBS 118.80) of Pythium aphanidermatum (Edson) Fitzp. for the ITS (761 bp), TUB2 (583 bp), and COXII (547 bp). Five isolates (2sp, 3sp, ND2-4sp, D3-4sp, and ND3-3sp) were 99.87%, 100%, and 99% identical to the reference strain (CBS 254.70) of Pythium myriotylum Drechsler for the ITS (762 bp), TUB2 (602 bp), and COXII (556 bp), respectively. Phylogenetic analysis of Pythium isolates inferred from concatenated sequences of the three genes (LéVesque and De Cock 2004; Villa et al. 2006) revealed that the same four isolates grouped with the neotype strain of P. aphanidermatum, and the five isolates clustered with the reference strain of P. myriotylum, each with a 100% bootstrap support. Morphological features of isolates ND3-3sp and sp01 were used for identification. Isolate ND3-3sp produced inflated lobulate sporangia and aplerotic and smooth oospores (16.3 to 25.1 um; n = 30) attached with three to five antheridia, consistent with identification as P. myriotylum. Isolate sp01 produced inflated lobulate sporangia and aplerotic and smooth oospores (17.0 to 24.0 um; n= 30) attached with a single intercalary antheridium, agreeing with the morphology of P. aphanidermatum (Van der Plaats-Niterink 1981). To investigate the pathogenicity of the nine Pythium isolates on spinach, 20 mycelial agar discs (4 mm in diameter) from a 2-day-old V8 culture of each isolate were used to induce sporangia and zoospores in 20 ml sterilized water at 25℃ with a 12 h light / dark regime. A 1.5 ml zoospore suspension (6 × 103 zoospores / ml) was dropped into BVB growth substrate of two spinach seedlings in 2-week-old at 25℃ with 12 h light / dark regime, resulting in symptoms resembling those observed in commercial nurseries at 7 days post-inoculation (dpi). Each Pythium isolate inoculated 20 seedlings in 10 cells of a planting tray. At 14 dpi, disease incidences were 95 to 100% for P. myriotylum isolates and 60 to 85% for P. aphanidermatum isolates, while control plants treated with water showed no symptoms. Re-isolated pathogens from the inoculated plants were morphologically identical to the inoculated isolates, completing Koch's postulates. Results of the pathogenicity assay, along with molecular and morphological identification, conclude that the root rot of spinach was caused by P. myriotylum and P. aphanidermatum. The two oomycetes were not formally documented to cause spinach diseases in Taiwan. Although P. myriotylum has been isolated from spinach (Wang et al. 2003), its pathogenicity to spinach was not documented worldwide. Root rot of spinach caused by P. aphanidermatum has been reported in the United States (Bates and Stanghellini 1984), Korea (Cho and Shin 2004), and Italy (Garibaldi et al. 2015). These pathogens thrive in humid and hot weather (Littrell and McCarter, 1970). Producing spinach in cooler weather or in a temperature-controlled environment may help prevent severe occurrence of the disease.

Biocontrol Potential of Trichoderma Ghanense and Trichoderma Citrinoviride toward Pythium aphanidermatum.

Pythium-induced damping-off of cucumber is a major constraint to cucumber production in different parts of the world. Although chemical fungicides are used for managing this disease, they have many drawbacks to the environment. The ability of the antagonistic fungi isolated from the rhizosphere and endosphere of Dactyloctenium robecchii and Moraea sisyrinchium in the control of soilborne pathogen Pythium aphanidermatum was inspected. Native Trichoderma isolates, Trichoderma ghanense and Trichoderma citrinoviride, were isolated from plant stem and soil samples collected from Al-Seeb, Oman. Using a dual culture technique, the antagonistic activity of the fungal isolates against P. aphanidermatum was examined in vitro. Among Trichoderma isolates, T. ghanense was more efficient in restraining the mycelial growth of P. aphanidermatum, causing an inhibition percentage of 44.6%. Further, T. citrinoviride induced significantly lower cessation of P. aphanidermatum mycelial growth (31.3%). Microscopic and electrolyte leakage inspection of the pathogen mycelia depicted extreme morphological malformations in their mycelium, which can be attributed to the antifungal metabolites of antagonists. Greenhouse studies demonstrated the effectivity of T. ghanense in controlling Pythium damping-off of cucumber plants, where the number of surviving plants was over 90% when the biocontrol agents were used compared to 0 in the control plants. Furthermore, treatment of the plants with the antagonists promoted growth characteristics of plants compared to uninoculated plants. This included improvements in shoot and root lengths, leaf length and width, and dry weight. These findings suggest that T. ghanense and T. citrinoviride can be developed as alternatives to synthetic chemical fungicides to manage soilborne pathogens of cucumber. This research is also the first to clarify the biocontrol ability of T. citrinoviride and T. ghanense against cucumber damping-off caused by P. aphanidermatum.

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.

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.

Effect of Infection Timing by Four Pythium spp. on Soybean Damping-Off Symptoms with and Without Cold Stress.

Pythium spp. cause damping-off of soybean, especially when soil conditions at or shortly after planting are cool and wet. Soybean planting dates continue to shift to earlier dates, so germinating seed and seedlings are exposed to periods of cold stress at a time which favors infection by Pythium, and seedling disease occurs. The objective of this study was to assess infection timing and cold stress on soybean seedling disease severity caused by four Pythium spp. prevalent in Iowa, namely P. lutarium, P. oopapillum, P. sylvaticum, and P. torulosum. Each species was used individually to inoculate soybean cultivar 'Sloan' using a rolled towel assay. Two temperature treatments (continuous 18°C [C18]; a 48-h cold stress period at 10°C [CS]) were applied. Soybean seedling age was divided into five growth stages (GS1 to GS5). Root rot severity and root length were assessed at 2, 4, 7, and 10 days after inoculation (DAI). At C18, root rot was greatest when soybean was inoculated with P. lutarium or P. sylvaticum at GS1 (seed imbibes water) and with P. oopapillum or P. torulosum at GS1, GS2 (radicle elongation), and GS3 (hypocotyl emergence). After CS, soybean susceptibility to P. lutarium and P. sylvaticum was reduced compared to C18 for inoculation at all GSs except GS5 (unifoliate leaf emergence). Conversely, root rot by P. oopapillum and P. torulosum was greater after CS compared to C18. Data from this study demonstrate that greater root rot, and consequently more damping-off, is likely if infection occurs at early germination stages before seedling emergence.

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.

Efficient Role of Endophytic Aspergillus terreus in Biocontrol of Rhizoctonia solani Causing Damping-off Disease of Phaseolus vulgaris and Vicia faba.

The wide spread of plant pathogens affects the whole world, threatening national food security. Various fungi including Rhizoctonia solani induce the fungal disease damping-off that negatively affects plant seedlings' growth. Recently, endophytic fungi are used as safe alternatives to chemical pesticides that harm plant and human health. Here, an endophytic Aspergillus terreus was isolated from Phaseolus vulgaris seeds to control damping-off diseases by improving the defense system in Phaseolus vulgaris and Vicia faba seedlings. Endophytic fungus was morphologically and genetically identified Aspergillus terreus, and it is deposited in GeneBank under accession OQ338187. A. terreus demonstrated antifungal efficacy against R. solani with an inhibition zone at 22.0 mm. Moreover, the minimum inhibitory concentrations (MIC) of ethyl acetate extract (EAE) of A. terreus were between 0.3125 and 0.625 mg/mL to inhibit R. solani growth. Precisely 58.34% of the Vicia faba plants survived when A. terreus was added compared with the untreated infected (16.67%). Similarly, Phaseolus vulgaris achieved 41.67% compared to the infected (8.33%). Both groups of treated infected plants showed reduced oxidative damage (reduced Malondialdehyde and hydrogen peroxide levels) as compared to untreated infected plants. Reduced oxidative damage was correlated with the increase in photosynthetic pigments and the antioxidant defense system including polyphenol oxidase, peroxidase, catalase, and superoxide dismutase enzyme activities. Overall, the endophytic A. terreus can be considered an effective tool to control the suppression of Rhizoctonia solani in legumes, especially Phaseolus vulgaris and Vicia faba, as an alternative to synthetic chemical pesticides that harm the environment and human health.

Comparative Study of Three Biological Control Agents and Two Conventional Fungicides against Coriander Damping-off and Root Rot Caused by Rhizoctonia solani.

The in vitro and in vivo efficacy of three biocontrol agents, Trichoderma viride, Pseudomonas fluorescence, and Bacillus subtilis, were tested against Rhizoctonia solani (AG-4) infection compared to two conventional fungicides (Rizolex-T 50%wettable powder and Amistar 25%). Antifungal enzyme activity was assayed in the culture filtrate of the biocontrol agents. The impact of the tested biocontrol agents on the induction of the coriander immune system was investigated against R. solani by assessing the resistance-related enzymes and compounds in biocontrol agent-treated plants compared with the control. The obtained results revealed that all tested biocontrol agents significantly reduced the linear growth of R. solani, and T. viride recorded the highest inhibition percentage. This could be linked to the ability of T. viride to produce higher activities of antimicrobial enzymes, i.e., cellulase, chitinase, and protease, compared to P. fluorescence and B. subtilis. Applying the tested biocontrol agents significantly alleviated pre- and post-emergence damping-off and root rot/wilt diseases of infected coriander compared with untreated plants. The tested biocontrol agents exhibited significantly higher germination percentage and vigor index of the coriander than the tested fungicides. The tested biocontrol agents significantly minimized the reduction of photosynthetic pigments induced by R. solani. In addition, the results showed a significant increase in enzymes/molecules (i.e., phenylalanine, catalase, peroxidase, catalase, superoxide dismutase, phenylalanine ammonia-lyase, phenolics, ascorbic acids, and salicylic acid) involved directly and indirectly in coriander resistance to R. solani. The principal component analysis of the recorded data recommended the role of the high accumulation of oxidative parameters (hydrogen peroxide and lipid peroxidation) and the inhibition of phenolic compounds in the downregulation of coriander resistance against R. solani. The heatmap analysis results revealed that biocontrol agents, especially Trichoderma, enhanced the resistance against R. solani via the stimulation of salicylic acid, phenolics, and antioxidant enzymes. Overall, the data recommended the efficacy of biocontrol agents, especially T. viride, against R. solani infecting coriander plants, which could be an efficient and a safer alternative to conventional fungicides.

Successional effects of cover cropping and deep tillage on suppression of plant-parasitic nematodes and soilborne fungal pathogens.

BACKGROUND: Cover crops can suppress soilborne nematodes and fungal pathogens by serving as a poor host to pathogens and producing allelopathic compounds. Yet, cultural practices can influence their effectiveness. Cover crop and weedy fallow rotations and their interactions with deep tillage were evaluated from 2019 to 2021 in a three-season vegetable cropping system (spring tomato, fall squash, and winter cabbage) for their suppressive effects on soilborne diseases. Experimental plots were arranged in a split-plot 2 × 4 factorial design in randomized complete blocks. Whole-plot tillage treatments were shallow-tilled or deep-tilled. Subplots had two factors of crop rotations: rotation type (cover crop [spring or fall sunn hemp or winter rye] or weedy fallow) and rotation season. RESULTS: Independent of tillage practice, sunn hemp and weedy fallow reduced population density and root galling severity of root-knot nematode (Meloidogyne incognita) for the first subsequent vegetable compared to the all-vegetable rotation (P < 0.05) but had little effect on fungal pathogens. Fall sunn hemp had higher plant biomass and reduced gall severity for the second subsequent vegetable. Spring and fall sunn hemp improved vegetable yields. Winter rye only reduced ring nematodes (Mesocriconema spp.) population density in the first subsequent vegetable. Deep tillage reduced incidence of fungal pathogens of Rhizoctonia solani and Sclerotinia sclerotiorum, and population density of stubby-root nematode (Nanidorus minor). CONCLUSION: Sunn hemp is effective in suppressing M. incognita, whereas deep tillage can be used to suppress R. solani, S. sclerotiorum, and N. minor in vegetable production systems. © 2023 Society of Chemical Industry.

Fusarioid community diversity associated with conifer seedlings in forest nurseries across the contiguous USA.

Introduction: Fusarioid fungi that cause damping-off and root diseases can result in significant losses to conifer crops produced in forest nurseries across the USA. These nurseries are vital to reforestation and forest restoration efforts. Understanding the diversity of Fusarioid fungi associated with damping-off and root diseases of conifer seedlings can provide an approach for targeted management techniques to limit seedling losses and pathogen spread to novel landscapes. Methods: This study identifies 26 Fusarium spp. (F. acuminatum, F. annulatum, F. avenaceum, F. brachygibbosum, F. clavus, F. commune, F. cugenangense, F. diversisporum, F. elaeagni, F. elaeidis, F. flocciferum, F. fredkrugeri, F. fujikuroi, F. grosmichelii, F. ipomoeae, F. lactis, F. languescens, F. luffae, F. odoratissimum, F. oxysporum, F. queenslandicum, F. redolens, F. torulosum, F. triseptatum, F. vanleeuwenii, & F. verticillioides), 15 potential species within Fusarium and Neocosmospora species complexes (two from F. fujikuroi species complex, nine from F. oxysporum species complex, three from F. tricinctum species complex, and one from Neocosmospora species complex), and four Neocosmospora spp. (N. falciforme, N. metavorans, N. pisi, & N. solani) and associated host information collected from conifer-producing nurseries across the contiguous USA. Results: Phylogenetic analyses identified Fusarioid fungi haplotypes that were associated with 1) host specificity, 2) localization to geographic regions, or 3) generalists found on multiple hosts across diverse geographic regions. Discussion: The haplotypes and novel species identified on conifer seedlings should be considered for further analysis to determine pathogenicity, pathogen spread, and assess management practices.

Synthesis of bis(ylidene) cyclohexanones and their antifungal activity against selected plant pathogenic fungi.

Botrytis cinerea, Rhizoctonia solani and Hemileia vastatrix are three species of phytopathogenic fungi behind major crop losses worldwide. These have been selected as target models for testing the fungicide potential of a series of bis(ylidene) cyclohexanones. Although some compounds of this chemical class are known to have inhibitory activity against human pathogens, they have never been explored for the control of phytopathogens until now. In the present work, bis(ylidene) cyclohexanones were synthesized through simple, fast and low-cost base- or acid-catalyzed aldol condensation reaction and tested in vitro against B. cinerea, R. solani and H. vastatrix. bis(pyridylmethylene) cyclohexanones showed the highest activity against the target fungi. When tested at 200 nmol per mycelial plug against R. solani., these compounds completely inhibited the mycelial growth, and the most active bis(pyridylmethylene) cyclohexanone compound had an IC50 of 155.5 nmol plug-1. Additionally, bis(pyridylmethylene) cyclohexanones completely inhibited urediniospore germination of H. vastatrix, at 125 µmol L-1. The most active bis(pyridylmethylene) cyclohexanone had an IC50 value of 4.8 µmol L-1, which was estimated as approximately 2.6 times lower than that found for the copper oxychloride-based fungicide, used as control. Additionally, these substances had a low cytotoxicity against the mammalian Vero cell line. Finally, in silico calculations indicated that these compounds present physicochemical parameters regarded as suitable for agrochemicals. Bis(ylidene) cyclohexanones may constitute promising candidates for the development of novel antifungal agents for the control of relevant fungal diseases in agriculture.

Yield Loss and Integrated Disease Control of Rhizoctonia solani AG2-1 Using Seed Treatment and Sowing Rate of Oilseed Rape.

Rhizoctonia solani anastomosis group (AG) 2-1 is an ubiquitous soilborne pathogen causing severe damping-off of oilseed rape (OSR). In the absence of varietal resistance to AG2-1, there are limited methods for integrated disease management. The objectives of these field studies were to quantify yield losses due to AG2-1 and to determine the effectiveness of integrated control using sedaxane, fludioxonil, and metalaxyl-M applied as seed treatment on two OSR genotypes at a sowing rate of 40 (low) or 80 (high) seeds m-2. Crop assessments of green area index (GAI), vigor, and cabbage stem flea beetle (CSFB) Psylliodes chrysocephala damage were carried out at GS16, while pathogen DNA in soil was quantified using real-time PCR at GS32. Yield and seed weight losses of 41 and 18%, respectively, were associated with reduced establishment, GAI, vigor, and delayed development and flowering of OSR. Seed treatment reduced AG2-1 DNA in soil by 80%, resulting in a 94, 16, and 64% increase of establishment, thousand seed weight (TSW), and yield, respectively. Seed treatment also mitigated the effects of AG2-1 on delaying plant development, resulting in increased uniformity of crop flowering. OSR plants infected with AG2-1 suffered 27% more damage by the CSFB, indicating positive pathogen-pest interaction at the expense of the OSR host. Optimum control of AG2-1 infection was achieved by integrating low sowing rate and seed treatment. However, under dual pest and pathogen attack, high sowing rates should be combined with the use of seed treatment to mitigate seedling death and delayed development caused by AG2-1 and CSFB damage.

First Report of Seedling Damping-Off of industrial Hemp (Cannabis sativa L.) caused by seed-transmitted Alternaria rosae in Italy.

In the years 2020-2021 as part of the activity of the Campania region hemp fiber project, variety comparison trials were carried out on 7 hemp varieties among those relevant for bast fiber production. During the trials, in particular on the cv. Fibrante, a consistent problem was noted: a noticeable germination failure (80-90%) occurred during the emergence of seedlings. Therefore, experiments were conducted to ascertain the possible presence of seed-borne pathogens. Tests were carried out on 100 seeds that were surface disinfected with 2% sodium hypochlorite solution for 3 min, rinsed in sterile distilled water three times and dried on sterile filter paper. The seeds were plated on potato dextrose agar (PDA Oxoid™) amended with 100 mg L-1 of streptomycin sulphate, kept at 24°C in the dark and observed daily. Growing colonies were subcultured on PDA for 10 days and, subsequently, twenty purified fungal isolates were obtained by single spore isolation. Colonies of these isolates on PDA were initially grayish-white and then turned dark olive green with abundant cotton-like aerial hyphae. On potato carrot agar (PCA) medium, these isolates produced light brown and solitary conidiophore with septum. Conidia were obclavate or pyriform, brown, with 1-3 transverse septa and 0-3 longitudinal septa, and measured 12.5 to 28.5 × 5 to 15 µm (n=50). The morphological characteristics observed under the light microscope were consistent with that of Alternaria spp. (Simmons 2007). In order to characterize the representative isolate, total DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and 3 genes were PCR-amplified: the ITS spacer using the primer pair ITS1-ITS4 (White et al., 1990), the transcription elongation factor 1- using the primer pair EF1-983F/ EF1-2218R (Rehner and Buckley., 2005) and the RNA polymerase II second largest subunit (RPB2) using the primer pair RPB2-5F2/fRPB2-7cR (Sung et al 2007; Liu et al 1999). The size-expected amplicons were purified and sequenced at the BMR Genomics (Padova, Italy) and the resulting sequences were deposited in GenBank under the accession numbers ON556507, ON601003, ON601004. BLAST-n analysis revealed 98 to 99% nucleotide identity with some representative isolates of Alternaria rosae E.G. Simmons & C.F. Hill (KU375630.1, XM_046169884.1, XM_046168987.1). To fulfill Koch's postulates, 100 hemp-certified seed were disinfected as mentioned above, left to germinate on the water-agar to discard potentially infected seeds and finally sowed in sterile peat-soil mix (1:1 v/v). The inoculum consisted of 10 mL of 105 conidial suspension obtained by the representative isolate (Ar_H1). Negative control seeds were inoculated with sterile water. After 5-7 days 100% of inoculated seedlings showed weak germinative vigor with yellowing of the epicotyls and dark areas on the root. The tissue narrowed and turned necrotic with abundant white mycelium covering the entire seedling. Small pieces of necrotic roots were plated on PDA and the same Alternaria-like colonies grew in 10 days. DNA sequencing confirmed the presence of A. rosae. Alternaria spp. are fungi that produce a wide range of toxic metabolites, harmful to food safety in the food uses of the seed. This finding further highlights that the quality of the hemp seed must be considered as a priority aspect in the entire hemp supply chain. To the best of our knowledge, this is the first report of A. rosae as seed-borne fungus on hemp.

Formulation of biofungicides based on Streptomyces caeruleatus strain ZL-2 spores and efficacy against Rhizoctonia solani damping-off of tomato seedlings.

This work aims at exploring an antagonistic actinobacterial strain isolated from the roots of Ziziphus lotus in bioformulation processes and the biocontrol of Rhizoctonia solani damping-off of tomato seedlings. The strain Streptomyces caeruleatus ZL-2 was investigated for the principal in vitro biocontrol mechanisms and then formulated in three different biofungicides: wettable talcum powder (WTP), sodium alginate propagules (SAP) and clay sodium alginate propagules (CAP). Compared to a marketed control products (Serenade® and Acil 060FS®), the formulated biofungicides were investigated against the R. solani damping-off of tomato cv. Aïcha seedlings. The strain ZL-2 produced chitinases, cellulases, ß-1,3-glucanases, cyanhydric acid and siderophores. It also showed strong antagonistic effect on the mycelial growth of R. solani. Bioautographic and HPLC analysis revealed the production of a single or several co-migrating antifungal compounds. The biofungicide WTP presented an attractive biocontrol effect by significantly reducing the disease severity index (DSI) compared to untreated seeds. No significant differences were obtained compared to the chemical treatment with Acil 060FS®. The viability of spores and biocontrol efficacy of the WTP were confirmed after 1-year storage. Strain ZL-2 has never been reported in the bioformulation of active biofungicides against Rhizoctonia solani damping-off and this work opens up very attractive prospects in the fields of biocontrol and crop improvement.

Plant Growth-Promoting Rhizobacteria Promote Growth of Seedlings, Regulate Soil Microbial Community, and Alleviate Damping-Off Disease Caused by Rhizoctonia solani on Pinus sylvestris var. mongolica.

As the excessive use of chemical fertilizers harms organisms and adversely affects the soil environment, the replacement of chemical fertilizers with biological fertilizers has attracted widespread attention as an environmental protection strategy. In this study, the effects of rhizosphere bacteria inoculation on growth of Pinus sylvestris var. mongolica seedlings, soil parameters, soil microbial community structure, and the biocontrol of damping-off were studied by pot experiments. The results showed that all three rhizosphere bacteria (Pseudomonas chlororaphis, Pseudomonas extremaustralis, and Acinetobacter lwoffii A07) tested exhibited growth-promoting properties, such as the production of indole-3-acetic acid, hydrolase, siderophores, and hydrogen cyanide; nitrogen fixation; and phosphorus solubilization. The application of the three bacteria increased plant biomass, root structure, and nutrient content and also increased soil nutrient content and enzyme activity. Bacterial inoculation promoted the growth of beneficial bacteria and antagonistic bacteria by adjusting the physicochemical properties of the soil, thereby improving the bacterial community structure. Among the soil features, available nitrogen, total nitrogen, available potassium, and urease activity were the main influencing factors. In addition, it was also found that bacterial inoculation significantly increased the activities of plant superoxide dismutase, catalase, peroxidase, and other defense enzymes; enhanced plant disease resistance; effectively inhibited damping-off; and promoted plant growth. In summary, the application of three rhizosphere bacteria systematically affected the interaction between plants, soil parameters, and soil microbial communities. These results provide a basis for understanding how rhizosphere bacteria promote the growth of P. sylvestris var. mongolica, thereby offering a promising sustainable alternative to chemical fertilizers.

Cannabis Seedlings Inherit Seed-Borne Bioactive and Anti-Fungal Endophytic Bacilli.

Throughout the hundreds of millions of years of co-evolution, plants and microorganisms have established intricate symbiotic and pathogenic relationships. Microbial communities associated with plants are in constant flux and can ultimately determine whether a plant will successfully reproduce or be destroyed by their environment. Inheritance of beneficial microorganisms is an adaptation plants can use to protect germinating seeds against biotic and abiotic stresses as seedlings develop. The interest in Cannabis as a modern crop requires research into effective biocontrol of common fungal pathogens, an area that has seen little research. This study examines the seed-borne endophytes present across 15 accessions of Cannabis grown to seed across Western Canada. Both hemp and marijuana seedlings inherited a closely related group of bioactive endophytic Bacilli. All Cannabis accessions possessed seed-inherited Paenibacillus mobilis with the capacity to solubilize mineral phosphate. Additionally, seeds were found to carry genera of fungal isolates known to be Cannabis pathogens and post-harvest molds: Alternaria, Penicillium, Cladosporium, Chaetomium, Aspergillus, Rhizopus, and Fusarium. Thirteen seed-borne endophytes showed antibiotic activity against Alternaria, Aspergillus, Penicillium, and Fusarium. This study suggests both fungal pathogens and bacterial endophytes that antagonize them are vectored across generations in Cannabis as they compete over this shared niche.

Effect of Oligogalacturonides on Seed Germination and Disease Resistance of Sugar Beet Seedling and Root.

Oligogalacturonides (OGs) are a bioactive carbohydrate derived from homogalacturonan. The OGs synthesized in this study significantly inhibited the mycelial growth of Rhizoctonia solani AG-4HGI in vitro, even at a low concentration (10 mg/L). The seed vigor test demonstrated that the application of 50 mg/L OGs to sugar beet seeds significantly increased average germination percentage, germination energy, germination index, and seedling vigor index. The same concentration of OGs also improved the seedling emergence percentage of sugar beet when seeds were sown in soil inoculated with D2 and D31 isolates, respectively. The lesion diameter on mature sugar beet roots caused by R. solani AG-4HGI isolates D2 and D31 also decreased by 40.60% and 39.86%, respectively, in sugar beets roots first treated with 50 mg/mL OGs in the wound site, relative to lesion size in untreated/pathogen inoculated wounds. Sugar beet roots treated with 50 mg/mL OGs prior to inoculation with the D2 isolate exhibited up-regulation of the defense-related genes glutathione peroxidase (GPX) and superoxide dismutase (SOD) by 2.4- and 1.6-fold, respectively, relative to control roots. Sugar beet roots treated with 50 mg/mL OGs prior to inoculation with D31 exhibited a 2.0- and 1.6-fold up-regulation of GPX and SOD, respectively, relative to the control. Our results indicate that OGs have the potential to be used for the protection of sugar beet against R. solani AG-4HGI.

First Report of Globisporangium heterothallicum Causing Seedling Disease on Guayule in Arizona.

Guayule (Parthenium argentatum A. Gray) is known for producing low-allergenic latex that is used in high end rubber products for medical use such as rubber gloves, catheters, and condoms. Currently, there are growing efforts from tire industry to commercialize guayule for rubber production in Arizona. During May 2019, wilting and death of c. 25% of seedling plants were observed in direct-seeded guayule fields in central Arizona. Symptoms of root rots and hypocotyl constriction were observed on affected seedling plants. To identify the causal agent, four symptomatic plants were collected to isolate the putative pathogen. Small pieces of symptomatic root (2-5 mm) were surface sterilized in 0.6% sodium hypochlorite for 1 min, rinsed copiously in sterile distilled water, blotted dry, and plated on 10% clarified V8-PARP (Jeffers and Martin 1986). Four oomycete-like isolates with abundant hyphal swellings were purified by transferring tips of single hypha onto new 20% CV8 plates and incubating at 23°C for one week. Sporangia were formed abundantly, globose or lemon-shaped (average 20 ± 4 × 20 ± 4 µm, n = 15). Isolates did not produce oospores (heterothallic). Genomic DNA was extracted from the mycelia of two isolates using DNeasy Plant Pro Kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. The internal transcribed spacer (ITS) region of rDNA and mitochondrially encoded cytochrome c oxidase 1 (cox 1) gene were amplified with primers ITS1/ITS4 (White et al., 1990) and OomCoxI-Levup/OomCoxI-Levlo (Martin and Tooley, 2003; Robideau et al., 2011) and the resulting amplicons were sequenced (GenBank Accession No. OL514636 and OL539842). A BLASTn search of 808-bp amplicon (OL514636) revealed 100% match with ITS sequences MT039880 which was G. heterothallicum causing root and crown rot of pepper in Turkey. BLAST analysis of the 658-bp amplicon (OL539842) showed 99.39 % identity with the COX 1 sequence of G. heterothallicum from tomato in Australia (MT981128). To fulfill Koch's postulates, pathogenicity tests were conducted twice on 2-week-old 'Az 2' guayule plants grown in 1.9-liter pots filled with a steam-disinfested potting mix. Pots were placed in a plastic container and watered three times a week by flooding, to create waterlogged conditions. Plants were maintained in a greenhouse and fertilized weekly with a 20-20-20 fertilizer at 1mg/ml. Twenty plants in 5 pots (4 plants/pot) were challenged with a G. heterothallicum isolate by drenching pot with 50 ml of a 1×106 zoospore/ml suspension. Twenty plants in 5 pots, serving as a control, received each 50 ml of distilled water. Symptoms of wilting and water-soaked root rot, and plant death were observed 2 weeks afterward, whereas control plants remained asymptomatic. G. heterothallicum was reisolated from necrotic roots of inoculated plants but not from control plants. G. heterothallicum has been increasingly reported as a pathogen of damping-off or root and crown rot on hosts such as alfalfa in Minnesota (Berg et al., 2017), soybean in Pennsylvania (Coffua et al., 2016), spinach in Sweden (Larsson, 1994), corn in China (Gan, et al., 2010), pepper in Turkey (Dervis, et al., 2020). To our knowledge, this is the first report of G. heterothallicum causing guayule seedling diseases in the United States. The presence of broad-host-range pathogen G. heterothallicum suggests that new strategies are needed for managing this pathogen to increase stands in direct-seeded guayule production system.

Study on the Preparation and Effect of Tomato Seedling Disease Biocontrol Compound Seed-Coating Agent.

Tomato damping-off and root rot are the two most common diseases of tomatoes at the seedling stage. At present, biological compound seed-coating agents are gradually replacing chemical agents in preventing and controlling plant diseases and insect pests, regulating plant growth, and ensuring crop yields. In this study, five biocontrol bacteria (Bacillus amyloliquefaciens (Ba), Bacillus subtilis (Bs wy-1), Bacillus subtilis (WXCDD105), Pseudomonas fluorescens (WXCDD51), and Bacillus velezensis (WZ-37)), with broad antibacterial spectra were mixed with auxiliary factors (inactive components of seed-coating agent) after fermentation to compound a seed-coating agent. In this study, the formula for a compound seed-coating agent was selected through orthogonal experiment. Gaseous silica was used as a thickener, and gum arabic and sodium dodecylbenzene sulfonate were used as a film-forming agent and dispersant, respectively. The mass of fumed silica, gum arabic, sodium dodecylbenzene sulfonate, and pearlescent powder was 1.3 g, 1 g, 0.05 g, and 0.5 g, respectively. Adding gibberellin can improve the ability of seed-coating agents to promote seed germination and plant growth. This showed high efficiency in preventing and controlling seedling diseases and promoting seedling growth. After 6 days of inoculation with Pythium aphanidermatum, which caused tomato damping-off disease, the seedling mortality rate was 26.7% lower than that of the sterile water control, and 20% lower than that of carbendazim. After 21 days of inoculation with Fusarium sp., which caused tomato root rot disease, the seedling mortality rate was 44.31% lower than that of the control, and 22.36% lower than that of carbendazim. The plant height, stem diameter, root length, fresh weight, and dry weight of tomato seeds treated with biological compound seed-coating agent were significantly higher than that of the control. We tested the shelf life of the biological compound seed-coating agent, and found that the effect of seed germination and radicle growth did not decrease. This research provides information on the production technology and application of biological seed-coating agents in tomato production.

Pathogenicity of Bacillus Strains to Cotton Seedlings and Their Effects on Some Biochemical Components of the Infected Seedlings.

Pathogenicity of eight Bacillus strains to seedlings of four cotton cultivars was evaluated under greenhouse conditions. Each of the tested cultivars was individually treated with powdered inoculum of each bacterial strain. Untreated seeds were planted as control treatments in autoclaved soil. Effects of the tested strains on levels and activities of some biochemical components of the infected seedlings were also assayed. The biochemical components included total soluble sugars, total soluble proteins, total free amino acids, peroxidase, polyphenol oxidase, phenols, and lipid peroxidation. ANOVA showed that Bacillus strain (B) was a very highly significant source of variation in damping-off and dry weight. Cotton cultivar (V) was a nonsignificant source of variation in damping-off while it was a significant source of variation in dry weight. B × V interaction was a significant source of variation in damping-off and a nonsignificant source of variation in dry weight. Bacillus strain was the most important source of variation as it accounted for 59.36 and 64.99% of the explained (model) variation in damping-off and dry weight, respectively. The lack of significant correlation between levels and activities of the assayed biochemical components and incidence of damping-off clearly demonstrated that these biochemical components were not involved in the pathogenicity of the tested strains. Therefore, it was hypothesized that the pathogenicity of the tested strains could be due to the effect of cell wall degrading enzymes of pathogenic toxins. Based on the results of the present study, Bacillus strains should be considered in studying the etiology of cotton seedling damping-off.