Enhanced production of pinosylvin stilbene with aging of Pinus strobus callus and nematicidal activity of callus extracts against pinewood nematodes

Pinosylvin stilbenes are phenolic compounds mainly occurring in the Pinaceae family. We previously reported that the accumulation of two pinosylvin stilbene compounds, dihydropinosylvin methyl ether (DPME) and pinosylvin monomethyl ether (PME), in Pinus strobus trees was highly enhanced by infection with pine wood nematodes (PWNs: Bursaphelenchus xylophilus), and these two compounds showed strong nematicidal activity against PWNs. In this work, we established a system of pinosylvin stilbene (DPME and PME) production via the in vitro culture of P. strobus calli, and we examined the nematicidal activity of callus extracts. Calli were induced from the culture of mature zygotic embryos of P. strobus. Optimized growth of calli was obtained in 1/2 Litvay medium with 1.0 mg/L 2,4-D and 0.5 mg/L BA. DPME and PME accumulation did not occur in nonaged (one-month-old) calli but increased greatly with prolonged callus culture. The concentrations of DPME and PME in three-month-old dark-brown calli were 6.4 mg/g DW and 0.28 mg/g DW, respectively. The effect of methyl jasmonate treatment on the accumulation of DPME and PME was evaluated in cell suspension culture of P. strobus. However, the treatment appeared to show slight increase of DPME accumulation compared to callus browning. A test solution prepared from crude ethanol extracts from aged calli (three months old) containing 120 µg/ml DPME and 5.16 µg/ml PME treated with PWNs resulted in 100% immobilization of the adult PWNs and 66.7% immobilization of the juvenile PWNs within 24 h. However, nonaged callus extracts did not show any nematicidal activity against juvenile PWNs and showed less than 20% nematicidal activity against adult PWNs. These results indicate that pinosylvin stilbenes can be effectively produced by prolonged culture of P. strobus calli, can be isolated using simple ethanolic extraction, and are applicable as beneficial eco-friendly compounds with nematicidal activity against PWNs.

Influence of the composition of nutrient media on the productivity and biological activity of the strain of entomopathogenic fungus Beauveria bassiana

The productivity and biological activity of the entomopathogenic fungus Beauveria bassiana (F-145) strain during the liquid-phase cultivation on various substrates for developing a biopesticide in its native form was analyzed (the research was carried out in 2019). For submerged cultivation, by-products from dairy and beer production (milk whey and brewing spent grain liquor) were used as components of the nutrient medium with addition of diesel fuel (DF) and Tween-80 as inducers of biological activity. It has been established, that the productivity of the strain on industrial by-product substrates was 1.5-2 times higher than on the Czapek medium. A high yield of a mycelial biomass with a titer of 108 -1010 CFU/ml was shown in a 5-day suspension based on a mixture of milk whey and brewing spent grain liquor. The biological activity of the culture suspension of the strain was determined. It was shown that the nematicidal activity of Beauveria bassiana strain with regard to nematodes of the Rhabditis sp. was largely manifested in a suspension obtained on a mixed medium with the addition of inducers. Ninety per cent death at mobile nematode stages was registered within one or two days of test-organism incubation. A complex nutrient medium composition containing by-products and inducers contributed to the preservation of the biological activity of the strain. The strain nematicidal activity was established at the level of 67-80 per cent with a titer of 106 -107 CFU/ml when the suspension was stored for 67 days.

Nematicidal Activity of the Endophyte Serratia ureilytica against Nacobbus aberrans in Chili Plants (Capsicum annuum L.) and Identification of Genes Related to Biological Control

The genus Serratia is widely distributed in soil, water, plants, animals, invertebrates, and humans. Some species of this genus have antifungal, antibacterial, and nematicidal activity. In this work, the nematicidal activity of the endophytic strain of Serratia sp. in chili, Capsicum annuum L., is reported, where at a bacterial concentration of 4 × 109 cel/mL, the penetration of nematodes into the roots significantly decreased by 91 and 55% at 7 and 21 days after inoculation. This bacterial concentration also significantly decreased the number of galls, eggs, egg masses and reproduction factor produced by Nacobbus aberrans in Chili plants, with respect to the control where this bacterial strain was not applied. In the analysis of the genome of the strain, based on average nucleotide identity (ANI), the isolate could be affiliated to the species Serratia ureilytica. The size of the genome is 5.4 Mb, with a 59.3% content of GC. Genes related to the synthesis of chitinases, siderophores, proteases C, serralisins, hemolysin, and serrawettin W2 that have been reported for biocontrol of nematodes were identified in the genome. It is the first report of Serratia ureilytica with nematicidal activity. Based on these results of nematicidal activity, this strain can be evaluated in the field as an alternative in the biocontrol of Nacobbus aberrans in chili cultivation.

Nematicidal Activity of Cyclopiazonic Acid Derived From Penicillium commune Against Root-Knot Nematodes and Optimization of the Culture Fermentation Process

Among 200 fungal strains isolated from the soil, only one culture filtrate of Aspergillus flavus JCK-4087 showed strong nematicidal activity against Meloidogyne incognita. The nematicidal metabolite isolated from the culture filtrate of JCK-4087 was identified as cyclopiazonic acid (CPA). Because JCK-4087 also produced aflatoxins, six strains of Penicillium commune, which have been reported to be CPA producers, were obtained from the bank and then tested for their CPA productivity. CPA was isolated from the culture filtrate of P. commune KACC 45973. CPA killed the second-stage juveniles of M. incognita, M. hapla, and M. arearia with EC50–3 days 4.50, 18.82, and 60.51 μg mL–1, respectively. CPA also significantly inhibited egg hatch of M. incognita and M. hapla after a total of 28 days of treatment with the concentrations > 25 μg mL–1. The enhancement of CPA production by P. commune KACC 45973 was explored using an optimized medium based on Plackett–Burman design (PBD) and central composite design (CCD). The highest CPA production (381.48 μg mL–1) was obtained from the optimized medium, exhibiting an increase of 7.88 times when compared with that from potato dextrose broth culture. Application of the wettable power-type formulation of the ethyl acetate extract of the culture filtrate of KACC 45973 reduced gall formation and nematode populations in tomato roots and soils under greenhouse conditions. These results suggest that CPA produced by P. commune KACC 45973 can be used as either a biochemical nematicide or a lead molecule for developing chemical nematicides to control root-knot nematodes.

A new strain of Volutella citrinella with nematode predation and nematicidal activity, isolated from the cysts of potato cyst nematodes in China

Background Plant parasitic nematodes (PPNs) are responsible for causing many plant diseases and are extremely difficult to control at present. Currently, due to the negative effects of chemical agents on the environment and human health, the development of new biological pesticides has become an important part of plant nematode control. Nematophagous fungi refers to a class of fungi that kill plant nematodes. Notably, a large number of nematophagous fungi resources remain to be studied. The objective of our study was to use in vitro screening to identify nematophagous fungi and select strains that were highly active against nematodes, providing a primary research for the development and utilization of new nematophagous fungi. Results A new nematophagous fungal strain (GUCC2219) was isolated from cysts of possibly Globodera spp. and Heterodera spp., identified as Volutella citrinella. The hyphae of V. citrinella produced ring structures of variable size and exhibited predatory and nematicidal activity. The hyphal predation rates (in vitro) against three species of nematodes, Aphelenchoides besseyi, Bursaphelenchus xylophilus, and Ditylenchus destructor, averaged 59.45, 33.35, and 50.95%, respectively, while the fermentation broth produced by the fungus exhibited mortality rates of 100, 100, and 55.63%, respectively, after 72 h. Conclusion V. citrinella is a new strain with nematophagous properties, which are a novel discovery. At the same time, this is the first report of nematicidal and nematode predation activity in the genus Volutella.

Pathogenicity and Volatile Nematicidal Metabolites from Duddingtonia flagrans against Meloidogyne incognita

Plant parasitic nematodes, especially parasitic root-knot nematodes, are one of the most destructive plant pathogens worldwide. The control of plant root-knot nematodes is extremely challenging. Duddingtonia flagrans is a type of nematode-trapping fungi (NTF), which produces three-dimensional adhesive networks to trap nematodes. In this study, the pathogenicity and volatile organic compounds (VOCs) of the NTF D. flagrans against the plant root-knot nematode, Meloidogyne incognita, were investigated. The predatory process of D. flagrans trapping M. incognita was observed using scanning electron microscopy. Gas chromatography-mass spectrometry analysis of the VOCs from D. flagrans led to the identification of 52 metabolites, of which 11 main compounds were tested individually for their activity against M. incognita. Three compounds, cyclohexanamine, cyclohexanone, and cyclohexanol, were toxic to M. incognita. Furthermore, these three VOCs inhibited egg hatching of M. incognita. Cyclohexanamine showed the highest nematicidal activity, which can cause 97.93% mortality of M. incognita at 8.71 µM within 12 h. The number of hatched juveniles per egg mass after 3 days was just 8.44 when treated with 26.14 µM cyclohexanamine. This study is the first to demonstrate the nematicidal activity of VOCs produced by D. flagrans against M. incognita, which indicates that D. flagrans has the potential to biocontrol plant root-knot nematodes.