Developing Penicillium digitatum Management Strategies on Post-Harvest Citrus Fruits with Metabolic Components and Colonization of Bacillus subtilis L1-21

Citrus is among the most important plants in the fruit industry severely infected with pathogens. Citrus green mold caused by Penicillium digitatum is one of the most devastating diseases during post-harvest stages of citrus fruit. In this study, a potential endophyte Bacillus subtilis L1-21, isolated from healthy citrus plants, was assessed for its biocontrol activity against the pathogen P. digitatum. Based on an in vitro crosstalk assay, we suggested that B. subtilis L1-21 inhibits the pathogen with an inhibition zone of 3.51 ± 0.08 cm. Biocontrol efficacy was highest for the fermented culture filtrate of B. subtilis L1-21. Additionally, using GC-MS analysis, 13 compounds were detected in the extract of this endophyte. The culture filtrate in Landy medium could enlarge and deform pathogen spores and prevent them from developing into normal mycelium. Accordingly, the Landy culture filtrate of B. subtilis L1-21 was stable in the temperature range of 4–90 °C and pH of 3–11. Further, MALDI-TOF-MS for B. subtilis L1-21 detected surfactin, fengycin, bacillaene and bacilysin as potential antifungal compounds. GFP-tagged B. subtilis L1-21 easily colonized in citrus fruit peel and pulp, suggesting its role in eliminating the fungal pathogen. Altogether, it is highly expected that the production of antifungal compounds, and the colonization potential of B. subtilis L1-21 are required against the post-harvest P. digitatum pathogen on citrus fruit.

Aspergillus niger Culture Filtrate (ACF) Mediated Biocontrol of Enteric Pathogens in Wastewater

Robust control of pathogens in sewage facilitates safe reuse of wastewater rich in valuable nutrients for potential valorization through biological means. Aspergillus niger is widely reported in bioremediation of wastewater but studies on control of enteric pathogens in sewage are very sparse. So, this study aimed at exploring the antibacterial and nematicidal activity of A. niger culture filtrate (ACF). Antibacterial activity of ACF on enteric pathogens (Klebsiella pneumoniae, Pseudomonas aeruginosa, Vibrio cholerae, Salmonella enterica, Shigella dysenteriae, Escherichia coli, Staphylococcus aureus, Klebsiella variicola) was determined by spectrophotometric growth analysis, resazurin based viability assay and biofilm formation assay. ACF showed inhibition against all enteric pathogens except Pseudomonas aeruginosa. Nematicidal studies on Caenorhabditis elegans showed 85% egg hatch inhibition and 52% mortality of L1 larvae. Sewage treatment with ACF at 1:1 (v/v) showed 2–3 log reduction in coliforms, Klebsiella, Shigella, Salmonella, S. aureus and Vibrio except Pseudomonas, indicating significant alteration of complex microbial dynamics in wastewater. Application of ACF can potentially be used as a robust biocontrol strategy against infectious microbes in wastewater and subsequent valorization by cultivating beneficial Pseudomonas.

OPTIMIZATION OF IN VITRO SELECTIVE MEDIA FOR SELECTING ANTHRACNOSIS-RESISTANT FLAX CELLS

Цель исследований – оптимизация селективных сред для проведения отбора in vitro каллусных клеток льна, устойчивых к культуральному фильтрату штаммов возбудителя антракноза и создание in vitro новых генотипов, устойчивых к болезни. В результате исследований уточнен состав культурального фильтрата штаммов антракноза. Выявлено, что токсичность культуральных фильтратов не зависела от вирулентности используемых штаммов – более токсичными оказались культуральные фильтраты штаммов 784 (сильновирулентного) и 780 (средневирулентного) (загнивание и отмирание первичных корешков на 5 сутки наблюдали у 67 – 88% проросших семян), менее токсичны – штаммы 793 (сильновирулентный) и 788 (слабовирулентный) (на 5 сутки загнивание и отмирание первичных корешков отмечено у 9 – 15% проросших семян). Установлено, что морфогенные очаги формировались активнее у генотипов, морфогенный каллус которых переносили на среду с аналогичной или более высокой концентрацией культурального фильтрата. Показано, что на 14 сутки во втором пассаже с большей частотой формировались морфогенные каллусы, почки и побеги при использовании в первом и втором пассажах селективной среды, содержащей культуральный фильтрат в концентрации 40 мл/л, или в первом пассаже – 40 мл/л, а во втором – 44 мл/л. Выделены генотипы, сохраняющие устойчивость к антракнозу в течение трёх поколений на уровне 50 – 60%: НО-78 х Ленок, HJI-103-2 х Ленок, НЛ-40-1 х Ленок, HЭ-38 х Росинка, НЭ-36 х Ленок, НЭ-17 х Ленок, HЭ-16-2 х Росинка. Research objective – optimization of selective media for in vitro selection of flax callus cells resistant to culture filtrate of anthracnose pathogen strains and in vitro creation of new disease-resistant genotypes. As a result of the research, the composition of the culture filtrate of anthracnose strains was clarified. It was revealed that the toxicity of cultural filtrates did not depend on the virulence of the strains used - cultural filtrates of strains 784 (highly virulent) and 780 (medium virulent) turned out to be more toxic (decay and death of primary roots on day 5 was observed in 67 - 88% of germinated seeds), less toxic - strains 793 (strongly virulent) and 788 (weakly virulent) (on the 5th day, decay and death of primary roots was noted in 9-15% of germinated seeds). It was found that morphogenic foci were formed more actively in genotypes, the morphogenic callus of which was transferred to a medium with a similar or higher concentration of the culture filtrate. It was shown that on the 14th day in the second passage, morphogenic callus, buds and shoots were formed with a greater frequency when using in the first and second passages a selective medium containing a culture filtrate at a concentration of 40 ml/l, or in the first passage - 40 ml/l, and in the second - 44 ml/l. Genotypes were identified that retain resistance to anthracnose for three generations at a level of 50 - 60%: NO-78 x Lenok, HJI-103-2 x Lenok, NL-40-1 x Lenok, NE-38 x Rosinka, NE-36 x Lenok, NE-17 x Lenok, NE-16-2 x Rosinka.

Investigations on Biosuppression of Wilt Disease in Tomato Using Cell-Free Culture Filtrate of Phytopathogenic Fusarium oxysporum f.sp. lycopersici

Potency of cell-free culture filtrate of pathogenic Fusarium oxysporum f.sp. lycopersici as systemic defense inducer against wilt disease in tomato was tested by treating seedlings with the filtrate before pathogen challenge. Infective conidia and the cell-free culture filtrate were prepared from F. oxysporum (Sensu lato) previously isolated from wilt diseased affected plants. Growth relations of the isolates were characterized under ambient temperature (25 ± 2°C) and viability of the infective conidia of the two isolates were evaluated. Treatments were done as follows: (a) treatment of seedlings with infective conidia only (Treatment-A) (b) culture filtrate treatment only (Treatment-B) (c) treatment with culture filtrate followed by inoculation with infective conidia (Treatment-C) and (d) the control which consisted of plants sprayed with sterile distilled water only (Treatment-D). Effects of fungal conidia on tomato seed germination as well as some agronomic characters of the tomato plants and disease incidence under treatments A, B, C and D were evaluated. The growth and sporulation rates of the F. oxysporum F-isolate-1 were 12.1 mm day-1 and 6.5 x 104 conidia cm-2 colony area respectively and the values for the second isolate, F-Isolate 2 were 6.1 mm day-1 and 5.4 x 104 conidia cm-2 colony area. The germination rates of the infective conidia of F-isolate 1 and F-Isolate 2 after 24 hours incubation period at ambient temperature were 70% and 85% respectively. Treatment of tomato seeds with conidia suspension containing 1.0 x 106 had no statistically significant effects on seed germination [F(2,12)=0.148, P=0.64]; the mean percentage germination of the seeds treated with F-Isolate 1, F-Isolate 2 conidia and the control were 52%,46% and 52% respectively after five days. In the Treatment-A (Infective conidia only), F-Isolate 1 and F-isolate 2 caused 100% and 80% wilt of the plant populations at 7 weeks post-inoculation, when the tomato plants were 9 weeks old and no wilt was recorded in the control. There was no wilt recorded under Treatment-B and Treatment-C also, but there was reduced flowering and the mean percentage number of leaves showing chlorosis were significantly higher than the control [F (4, 192) =6.641, P=0.001]. When the plants were 10 weeks old, withered branches at the lower vegetal strata and the senescent leaves in the control were comparable with that recorded under Treatment-B and Treatment-C (F (4, 159) =3.563, P=0.08].

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.

Efectele genetice implicate în răspunsul grăului comun la filtratul de cultură Drechslera sorokiniana (SACC.) subram

Helminthosporiosis caused by the fungus Drechslera sorokiniana (Sacc.) causes significant crop and quality losses to Triticum aestivum L. in agroecological conditions with extreme humidity. Increasing the resistance is considered the most cost-effective and sustainable approach to disease control. The aim of this study was to determine the genetic effects involved in the inheritance of resistance, using the ge-netic model of character reproduction in descendants of wheat. Generations F1, F2, BCP1 and BCP2, de-scended from the mutual crossing of the parents Basarabeanca / Moldova 30 and Moldova 30 / Moldova 3 (P1 and P2) were evaluated for the response of callus characters to the action of D. sorokiniana culture filtrate on the medium Murashige Skoog. Fungal metabolites have decreased the effects of gene actions and epistatic interactions, but also their variance. The phenomenon corresponds to the decrease of callus indices. A great importance for the heredity of the character of the surface of the callus manifested the epistatic effects of additive-dominant (ad) type. In the case of callus biomass comparable to the mean val-ues were the a actions, but also the ad and dd epistatic effects. The predominant involvement of epistatic effects indicates the need for resistance selections to helminthosporiosis in late generations of wheat.

Growth and Characteristics of Two Different Epichloë sinensis Strains Under Different Cultures

In the present study, two Epichloë sinensis endophyte strains isolated from different Festuca sinensis ecotypes were inoculated on potato dextrose agar (PDA) and potato dextrose broth (PDB) media with or without (control) exogenous additives. After 4weeks of growth, the growth (colony diameter, hyphal diameter, and mycelial biomass) and other characteristics (pH and antioxidant capacity of culture filtrate, mycelial ion contents, and hormone contents) were measured. The results showed that the culture conditions had significant effects (p<0.05) on the hyphal diameter, mycelial biomass, and hormone content of the two strains. The mycelial biomass of the two strains in PDB was significantly higher (p<0.05) than that on PDA. Except for strain 1 with indole-3-acetic acid (IAA) treatment and strain 84F with control and VB1 treatments, the hyphal diameter of the two strains in PDB under the other treatments was significantly higher (p<0.05) than that on PDA. In most cases, the IAA, cytokinins (CTK), abscisic acid (ABA), and gibberlic acid (GA) contents in the mycelia on PDA of the two strains were significantly higher (p<0.05) than those in PDB. The two E. sinensis strains exhibited significantly different performances (p<0.05) under the five treatments. The indices, including colony diameter, mycelial biomass, scavenging ability of superoxide anion radicals and hydroxyl radicals, pH of culture filtrate, ion contents, hyphal diameter, and IAA, CTK, GA, and ABA contents were significantly different (p<0.05) between the two strains, although the performance was inconsistent. Exogenous additives had significant effects (p<0.05) on the performance of the two E. sinensis strains. Indole-3-acetic acid and VB1 treatments significantly promoted (p<0.05) the growth of the two strains on both PDA and PDB. Indole-3-acetic acid treatment also significantly increased the hyphal diameters of the two strains in PDB (p<0.05). Indole-3-acetic acid and VB1 treatments significantly reduced (p<0.05) the antioxidant ability of these two strains in PDB. NaCl and ZnCl2 treatments had significant inhibitory effects (p<0.05) on fungal growth and promotion effects on the antioxidant ability of the two strains. The treatments also had significant effects (p<0.05) on hyphal diameters and ion and hormone contents, although the effects varied with different indices.