Sebastian Kiewnick

In vitro activity of Bacillus firmus against the burrowing nematode Radopholus similis, the root-knot nematode Meloidogyne incognita and the stem nematode Ditylenchus dipsaci

Abstract Antagonistic bacteria have been repeatedly shown to be promising microorganisms for the biological control of sedentary and migratory endoparasitic nematodes. Depending on the bacteria involved, the mechanisms of action include: obligate parasitism, reduction in penetration, growth inhibition due to competition for nutrients and antibiosis associated with bioactive metabolites. In the present studies, the mode of action of the antagonist bacteria Bacillus firmus, isolated from a bionematicide, was evaluated. Significant rates of paralysis and mortality were detected after incubation of three nematode species in low concentrations of the pure culture filtrates following removal of the bacterial cells. The same culture filtrates also significantly reduced hatching of Meloidogyne incognita. Pure bacterial cell suspensions added to sand also reduced survival of R. similis in bioassays by 41% over the controls. The mode-of-action responsible for nematode paralysis and mortality was therefore demonstrated to be closely associated with the production of bioactive compounds secondary metabolites by the bacteria.

Evaluation of Paecilomyces lilacinus strain 251 for the biological control of the northern root-knot nematode Meloidogyne hapla Chitwood

The egg-pathogenic fungus Paecilomyces lilacinus strain 251 (PL215) was investigated under different temperature regimes for biocontrol activity against the northern root-knot nematode Meloidogyne hapla . Biocontrol activity by PL251 was affected by temperature and initial nematode inoculum density. At 20/23°C (night/day) control efficacy reached 71% at a high inoculum density of 10 000 eggs and juveniles (E + J) per 500 cm 3 compared to 65% control at the lower inoculum density (5000 E + J/500 cm 3 ). By contrast, average efficacy was 5% and 46% at low and high inoculum levels, respectively, when temperatures were between 19°C and 21°C (night/day). A significant interaction between the factors inoculum density and treatment with PL251 was observed at low temperatures, when only few M. hapla galls and egg masses developed after inoculation with 5000 E + J/500 cm 3 . When the temperature was favourable for the fungal parasite (25°C), efficacy reached 90%. In glasshouse experiments with unfavourable temperatures for M. hapla development (25°C-33°C), tomato plants repeatedly treated with PL251 showed significantly higher fruit yield (23-102%).

Efficacy of abamectin seed treatment on Pratylenchus zeae, Meloidogyne incognita and Heterodera schachtii

The aim of this study was to test the effect of various concentrations of abamectin on the reduction of early root penetration of three nematode species and on plant growth, when applied as a seed treatment on maize, cotton and sugar beet. The study revealed that penetration of Pratylenchus zeae was reduced more than 80% in maize at a dose of 1.0 mg a.i. seed−1. The number of galls caused by Meloidogyne incognita race 3 in cotton was also reduced more than 80% with 0.1 mg a.i. seed−1. Penetration of Heterodera schachtii in sugar beets was reduced over 60% when seeds were treated at a concentration of 0.3 mg a.i. seed−1. Root length, as well as shoot and root weights in all plants treated with abamectin were not significantly different from controls. Our investigation determined that using abamectin as a seed treatment is an effective way of reducing early root infestation of the three different nematodes at low concentrations in a variety of crops and does not negatively affect plant growth.ZusammenfassungZiel der vorliegenden Arbeit war es, den Einfluss verschiedener zur Saatgutbehandlung eingesetzter Abamectin-Konzentrationen auf Befall von Mais, Baumwolle und Zuckerrübe mit pflanzenparasitären Nematodenarten zu untersuchen sowie mögliche Nebenwirkung auf das Pflanzenwachstum zu ermitteln. Die Untersuchungen zeigten, dass bei einer Saat-gutbehandlung mit Abamectin mit einer Konzentration von 1.0 mg a.i. seed−1 die Eindringung von Pratylenchus zeae in Mais über 80% reduziert wurde. Im Falle von Baumwollsaat-gut, welches mit einer Abamectin-Konzentration von 0.1 mg a.i. seed−1 behandelt wurde, betrug die Wirksamkeit gegen-über Meloidogyne incognita (Rasse 3) ebenfalls 80%, bei Zuckerrübensaatgut, behandelt mit einer Abamectin-Konzen-tration von 0.3 mg a.i. seed−1, wurde eine Wirksamkeit gegen-über Heterodera schachtii von 60% festgestellt. Wurzellänge als auch die Spross- bzw. Wurzelgewichte von Pflanzen, deren Saatgut mit Abamectin behandelt wurde, unterschieden sich nicht signifikant von denen unbehandelter Pflanzen. Die Untersuchungen verdeutlichten, dass eine Saatgutbehand-lung mit Abamectin für die praktische Anwendung geeignet ist. Eine hohe Wirksamkeit in verschiedenen Kulturen ohne negative Auswirkungen auf das Pflanzenwachstum wurde festgestellt.

Endophytic bacteria from Ethiopian coffee plants and their potential to antagonise Meloidogyne incognita

Summary – Endophytic bacteria were isolated from coffee roots in Ethiopia and identified by Fatty Acid Methyl Ester-Gas Chromatography (FAME-GC). A total of 201 and 114 endophytic bacteria were isolated and identified during the wet and dry seasons, respectively. The most abundant genera were Pseudomonas, Bacillus, Agrobacterium, Stenotrophomonas and Enterobacter. Population densities were higher during the wet season than the dry season ranging from 5.2 × 10 3 to 2.07 × 10 6 cfu (g fresh root weight) −1 . Culture filtrates of the bacterial isolates showed nematicidal effects of between 38 and 98%. The most active strains were Agrobacterium radiobacter, Bacillus pumilus, B. brevis, B. megaterium, B. mycoides, B. licheniformis, Chryseobacterium balustinum, Cedecea davisae, Cytophaga johnsonae, Lactobacillus paracasei, Micrococcus luteus, M. halobius, Pseudomonas syringae and Stenotrophomonas maltophilia. Bacillus pumilus and B. mycoides were most effective in reducing the number of galls and egg masses caused by M. incognita by 33 and 39%, respectively.

Persistence of the nematophagous fungus Paecilomyces lilacinus strain 251 in soil under controlled conditions

Abstract The persistence of the nematophagous fungus Paecilomyces lilacinus Samson strain 251 (PL251) and the effect of application rate, substrate type, as well as the presence of the nematode host on its dynamics after application to the soil were investigated under controlled conditions. In all experiments, increase of P. lilacinus colony forming units after application was not found. In contrast, a gradual decline in fungal densities over time was observed. Application rate had no significant effect on the dynamics of the fungal population. Likewise, P. lilacinus density decline in soil was not significantly affected by the presence of the nematode host. Substrate type had a significant effect on P. lilacinus persistence in soil. The fungal agent persisted longer in silty loam and clay soil, with reduced persistence when sand was added to field soil. Conversely, when organic substrate was added to pure sand, persistence was significantly increased. Although persistence of fungal biocontrol agents in soil depends on various biotic and abiotic conditions, baseline data on persistence such as those reported in this study are helpful for biocontrol and environmental risk assessment and merit further study.

Detection of Heterodera schachtii infestation in sugar beet by means of laser-induced and pulse amplitude modulated chlorophyll fluorescence

Two glasshouse experiments with sugar beet cvs Penta and Macarena inoculated, respectively, with 0 or 1500 and 0, 500, 1000 or 1500 juveniles of Heterodera schachtii, were conducted to estimate the capability of laser-induced chlorophyll fluorescence (LIF) and pulse amplitude modulated (PAM) chlorophyll fluorescence techniques to detect H. schachtii infestation and to differentiate between infestation levels. Fluorescence and gas exchange parameters, nitrogen and chlorophyll content of sugar beet leaves were measured weekly after nematode inoculation. Sugar beet plants responded to H. schachtii infestation initially with a decrease in photosynthesis rate and later with a reduction in nitrogen uptake and chlorophyll concentration. At the early stages of nematode infestation, before visual symptoms were evident, infested sugar beet plants displayed increased fluorescence (F680, F740). Later stages of infection were accompanied by an increase in the F686/F740 ratio, ground fluorescence (Fo) and a decrease in photochemical efficiency (Fv/Fm) induced by degradation of leaf chlorophyll. Sugar beet plants infested with 500, 1000 or 1500 juveniles per 100 cm3 of soil did not differ either in their nitrogen and chlorophyll content or in photosynthesis and transpiration rate. The linear discrimination analysis based on the combination of PAM and LIF parameters resulted in 100% correct classification of control plants and high classification rates (60-100%) of the infested treatments on all the sampling dates. Whether the fluorescence technique will differentiate nematode densities under field conditions needs further study.

Effect of Meloidogyne incognita inoculum density and application rate of Paecilomyces lilacinus strain 251 on biocontrol efficacy and colonization of egg masses analyzed by real-time quantitative PCR.

The fungal biocontrol agent, Paecilomyces lilacinus strain 251 (PL251), was evaluated for its potential to control the root-knot nematode Meloidogyne incognita on tomato at varying application rates and inoculum densities. Conversely to previous studies, significant dose-response relationships could not be established. However, we demonstrated that a preplanting soil treatment with the lowest dose of commercially formulated PL251 (2 × 10(5) CFU/g soil) was already sufficient to reduce root galling by 45% and number of egg masses by 69% when averaged over inoculum densities of 100 to 1,600 eggs and infective juveniles per 100 cm(3) of soil. To determine the role of colonization of M. incognita egg masses by PL251 for biocontrol efficacy, a real-time quantitative polymerase chain reaction (PCR) assay with a detection limit of 10 CFU/egg mass was used. Real-time PCR revealed a significant relationship between egg mass colonization by PL251 and the dose of product applied to soil but no correlation was found between fungal density and biocontrol efficacy or nematode inoculum level. These results demonstrate that rhizosphere competence is not the key mode of action for PL251 in controlling M. incognita on tomato.

Effect of temperature on growth, germination, germ-tube extension and survival of Paecilomyces lilacinus strain 251

Abstract Paecilomyces lilacinus and in particular the commercial strain 251 has been intensively tested for biological control of plant parasitic nematodes. Since this species has been mentioned in a number of reports concerning infection of humans, the human health risk for Paecilomyces lilacinus strain 251 was investigated. The effects of time, temperature and growth medium on radial colony growth and germination were determined. Additionally, exposure to 36°C and its effect on germ-tube extension and on survival of conidia was evaluated. Radial growth was significantly affected by temperature, growth medium and their interaction. Optimum temperatures were between 24 and 30°C, but no growth was found at 36°C. Germination rate was significantly influenced by time, medium, temperature and their interactions. The optimum temperature range for germination was between 28 and 30°C. Formulated conidia were capable of germinating at 36°C. However, studies on germ-tube extension conducted at 36°C showed a delay in development for 28–49 h and no further germ-tube extension was found after exposure for 80–95 h. Slopes of survival curves were significantly influenced by the type of conidia tested. In general, conidia did not survive exposure to 36°C for 168 h. These experiments indicate the temperature conditions where the strain is likely to be active and provide supporting data for full environmental and health risk assessments of biocontrol fungi.

Interactions of Paecilomyces lilacinus strain 251 with the mycorrhizal fungus Glomus intraradices: Implications for Meloidogyne incognita control on tomato

Abstract The interactions of Paecilomyces lilacinus strain 251 with the arbuscular mycorrhizal fungus Glomus intraradices and their significance for the control of Meloidogyne incognita on tomato were investigated in greenhouse experiments. Application of P. lilacinus had no effect on the frequency and intensity of tomato root colonization by G. intraradices. Likewise, the decline of the nematophagous fungus densities after single application in soil was not affected by the presence of the mycorrhizal fungus. Single application of P. lilacinus, as pre-planting soil treatment, resulted in significant reduction of nematode damage. In contrast, mycorrhizal inoculation did not provide sufficient biocontrol. Combined application of the two agents did not enhance root protection compared to single treatments. Double treatment of mycorrhized seedlings with P. lilacinus, as seedling drench and pre-planting soil treatment, 4 and 1 week before transplanting, respectively, resulted in the highest reduction of the nematode damage. These results indicate the potential of the commercial P. lilacinus strain 251 and mycorrhiza for integration in nematode control strategies.

Effective concentration and range of activity of abamectin as seed treatment against root-knot nematodes in tomato under glasshouse conditions

The objectives of this study were to determine the efficacy, range of activity and effective concentrations (EC50 and EC80) of abamectin as a seed treatment of tomato against Meloidogyne incognita, M. arenaria and M. javanica. The study revealed that abamectin seed treatment at concentrations ranging between 0.3 and 1 mg a.s. seed−1 is highly effective against the three species of root-knot nematodes, retaining its efficacy in the soil for 8 weeks. The highest EC50 found was reached at 0.2 mg a.s. seed−1. The highest EC80 for the number of egg masses per g root in the three Meloidogyne species was attained at 0.51 mg a.s. seed−1. Abamectin as a seed treatment is promising for the control of root-knot nematodes in the field since only low amounts of active ingredient are required to give adequate protection in the most sensitive stages of tomato root growth and development.