Effects of Paecilomyces lilacinus protease and chitinase on the eggshell structures and hatching of Meloidogyne javanica juveniles

A serine protease and an enzyme preparation consisting of six chitinases, previously semi-purified from a liquid culture of Paecilomyces lilacinus strain 251, were applied to Meloidogyne javanica eggs to study the effect of the enzymes on eggshell structures. Transmission electron microscopic studies revealed that the protease and chitinases drastically altered the eggshell structures when applied individually or in combination. In the protease-treated eggs, the lipid layer disappeared and the chitin layer was thinner than in the control. The eggs treated with chitinases displayed large vacuoles in the chitin layer, and the vitelline layer was split and had lost its integrity. The major changes in the eggshell structures occurred by the combined effect of P. lilacinus protease and chitinases. The lipid layer was destroyed; the chitin layer hydrolyzed and the vitelline layer had lost integrity. The effect of P. lilacinus protease and chitinase enzymes on the hatching of M. javanica juveniles was also compared with a commercially available bacterial chitinase. The P. lilacinus protease and chitinase enzymes, either individually or in combination, reduced hatching of M. javanica juveniles whereas a commercial bacterial chitinase had an enhancing effect. Some juveniles hatched when the eggs were exposed to a fungal protease and chitinase mixture. We also established that P. lilacinus chitinases retained their activity in the presence of endogenous protease activity.     

Effects of culture filtrates from the nematophagous fungus Verticillium leptobactrum on viability of the root-knot nematode Meloidogyne incognita

Filtrates of three isolates of the nematophagous fungus Verticillium leptobactrum were evaluated for their nematicidal activity against the root-knot nematode Meloidogyne incognita. The filtrates inhibited egg hatching, with maximum toxicity observed for isolate HR21 at 50% (v:v) dilution, after 7 days exposure. Filtrates also inactivated second-stage juveniles (J2) at 10-50% dilutions. A scanning electron microscopy study of treated eggs showed severe alterations caused by the filtrate of isolate HR43 on M. incognita eggs, which appeared collapsed and not viable, suggesting the production of chitin-degrading enzymes or other active compounds.     

Potential of endophytic Streptomyces spp. for biocontrol of Fusarium root rot disease and growth promotion of tomato seedlings

Sixteen endophytic actinobacteria isolated from roots of native plants were evaluated for their antagonistic potential against soil-borne phytopathogenic fungi. Among them, three strong antagonistic isolates were selected and characterised for in vitro plant-growth-promoting and biocontrol traits, including production of hydrogen cyanide, indole-3-acetic acid and siderophores, chitinase and β-1,3-glucanase activities, and inorganic phosphate solubilisation. In all trials, the strain Streptomyces sp. SNL2 revealed promising features. The selected actinobacteria were investigated for the biocontrol of Fusarium oxysporum f. sp. radicis lycopersici and for growth promotion of tomato (Solanum lycopersicum L. cv. Aïcha) seedlings in autoclaved and non-autoclaved soils. All seed-bacterisation treatments significantly reduced the root rot incidence compared to a positive control (with infested soil), and the isolate SNL2 exhibiting the highest protective activity. It reduced the disease incidence from 88.5% to 13.2%, whereas chemical seed treatment with Thiram® provided 14.6% disease incidence. Furthermore, isolate SNL2 resulted in significant increases in the dry weight, shoot and root length of seedlings. 16S rDNA sequence analysis showed that isolate SNL2 was related to Streptomyces asterosporus NRRL B-24328^T (99.52% of similarity). Its interesting biocontrol potential and growth enhancement of tomato seedlings open up attractive uses of the strain SNL2 in crop improvement.     

Alginate films for delivery of root-knot nematode inoculum and evaluation of microbial interactions

A method was developed for utilizing alginate films to deliver inoculum into soil and evaluate microbial antagonistic activity against nematode eggs. Eggs of Meloidogyne incognita were harvested from galled tomato roots (Lycopersicon esculentum), surface disinfested, suspended in 2% (w/v) aqueous sodium alginate, and applied to 2.5 × 5.0 cm polyvinyl chloride coated fiberglass screens (1.5 mm² mesh size) at a uniform thickness of 0.5 mm. The alginate solution was gelled by dipping in 0.25 M CaCl₂. Films containing eggs were observed in vitro and egg development was evaluated. The number of immature eggs and eggs with first stage juveniles declined linearly over time while the number of empty eggs shells, and hatched juveniles increased over time, indicating that the alginate gel did not inhibit development and motility of M. incognita juveniles. In a greenhouse experiment using cucumber (Cucumis sativus) the number of galls g^-1 root was correlated with the number of eggs in alginate films placed in each pot at planting. Films containing M. incognita eggs were buried in field soil containing organic amendments, incubated, removed from soil, rinsed with water, and observed. The number of immature eggs in grids from soil amended with chitin or flax seed meal were lower than in untreated soil; percent parasitized eggs was also greater in films from amended soil than from untreated soil.     

Bio-control activity of Purpureocillium lilacinum strains in managing root-knot disease of tomato caused by Meloidogyne incognita

The potential of 24 indigenous isolates of Purpureocillium lilacinum (Paecilomyces lilacinus) (Thom) Samson collected from different agro-climatic zones of India was investigated against the root-knot nematode, Meloidogyne incognita. The studies were conducted in vitro (larvicidal, ovicidal and egg-parasitising capacity) and under naturally infested field conditions with selected strains. Repeated field trials were conducted with talc-based preparations of fungal strains at 10 kg ha^?1, which were applied mixed in farm yard manure (FYM) at 1.5 t ha^?1. Results (in vitro) showed that all tested isolates were capable to parasitise eggs, inhibit egg hatching and cause juvenile mortality of M. incognita at various levels. Based on the performance under in vitro studies, eight isolates (NDPL-01, ANDPL-02, SHGPL-03, HYBPL-04, AHDPL-05, PTNPL-06, SNGPL-07 and VNSPL-08) were re-tested to confirm the results. HYBDPL-04 was found causing highest mortality (80%), inhibition of egg hatching (90%) as well as parasitisation of M. incognita eggs (75%). Under field trials also, the best protection of root-knot disease of tomato (Lycopersicon esculentum L.), in terms of reduction of galls (61%) and reproductive factor (P_f/P_i (RF) = 0.2) was achieved through application of HYBDPL-04 + FYM compared to control and other tested isolates. It also enhanced marketable yield of tomato up to 43%. It is concluded that the HYBDPL-04 strain of P. lilacinum is highly effective for management of root-knot disease of tomato under naturally infested field conditions. It is the isolate which produced the maximum number of metabolites which were extracted through high pressure liquid chromatography.     

Water loss from eggs of domestic fowl and calcium status of hatchlings

Water balance in eggs of domestic fowl was manipulated by drilling holes (each 3 mm in diameter) through the calcareous layer into the air cell on day 10 of incubation. Water loss between days 0 and 18 averaged 6 g for eggs in the control group (no hole) but increased to 8, 12, and 15 g for eggs with 1, 2, or 3 holes, respectively. Hatching success was 79–87% for eggs with 0–2 holes through the eggshell but only 43% for eggs with three holes. Live mass of hatchlings declined as the number of holes drilled in the eggshell increased, but dry mass of carcasses was unaffected by the treatments. The quantity of Ca²⁺, Mg²⁺, and phosphorus in residual yolks and yolk-free carcasses of hatchlings was not influenced by the amount of water lost from eggs. Plasma Ca²⁺ and Mg²⁺ were elevated in hatchlings from eggs with high rates of water loss. The inability to regulate plasma Ca²⁺ may be a negative consequence of excessive water loss and could contribute to increased mortality of embryos.     

Purification and properties of a chitinase from Penicillium sp. LYG 0704

The chitinase producing Penicillium sp. LYG 0704 was procured from soil of the Chonnam National University crop field. The chitinase activity was detected after the first day which increased gradually and reached its maximum after 3 days of cultivation. The chitinase was purified from a culture medium by precipitation with isopropanol and column chromatography with Mono Q and Butyl-Sepharose. The molecular mass of chitinase was estimated to be 47 kDa by SDS–PAGE. Optimal pH and temperature were 5.0 and 40 °C, respectively. The N-terminal amino acid sequence of the enzyme was determined to be ¹AGSYRSVAYFVDWAI¹⁵. The fully cloned gene, 1287 bp in size, encoded a single peptide of 429 amino acids. BLAST search of the chitinase gene sequence showed similarity with chitinase of Aspergillus fumigatus Af293 chitinase gene (58%) and A. fumigatus class V chitinase ChiB1 gene (56%).     

Induction of defense-related proteins in tomato roots treated with Pseudomonas fluorescens Pf1 and Fusarium oxysporum f. sp. lycopersici

Pseudomonas fluorescens isolate Pf1 was found to protect tomato plants from wilt disease caused by Fusarium oxysporum f. sp. lycopersici. Induction of defense proteins and chemicals by P. fluorescens isolate Pf1 against challenge inoculation with F. oxysporum f. sp. lycopersici in tomato was studied. Phenolics were found to accumulate in bacterized tomato root tissues challenged with F. oxysporum f. sp. lycopersici at one day after pathogen challenge. The accumulation of phenolics reached maximum at the 5^th day after pathogen challenge. In pathogen-inoculated plants, the accumulation started at the 2^nd day and drastically decreased 4 days after the pathogen inoculation. Activities of phenylalanine ammonia-lyase (PAL), peroxidase (PO) and polyphenol oxidase (PPO) increased in bacterized tomato root tissues challenged with the pathogen at one day after pathogen challenge and activities of PAL and PO reached maximum at the 4^th day while activity of PPO reached maximum at the 5^th day after challenge inoculation. Isoform analysis revealed that a unique PPO1 isoform was induced and PO1 and PPO2 isoforms were expressed at higher levels in bacterized tomato root tissues challenge inoculated with the pathogen. Similarly, -1,3 glucanase, chitinase and thaumatin-like proteins (TLP) were induced to accumulate at higher levels at 3-5 days of challenge inoculation in bacterized plants. Western blot analysis showed that chitinase isoform Chi2 with a molecular weight of 46 kDa was newly induced due to P. fluorescens isolate Pf1 treatment challenged with the pathogen. TLP isoform with molecular weight of 33 kDa was induced not only in P. fluorescens isolate Pf1-treated root tissues challenged with the pathogen but also in roots treated with P. fluorescens isolate Pf1 alone and roots inoculated with the pathogen. These results suggest that induction of defense enzymes involved in phenylpropanoid pathway and accumulation of phenolics and PR-proteins might have contributed to restriction of invasion of F. oxysporum f. sp. lycopersici in tomato roots.     

Efficiency of Trichoderma harzianum as a biocontrol agent in combination with humate and chitosan against Meloidogyne incognita on tomato

Abstract

A pot trial was conducted to estimate the role of Trichoderma harzianum alone or in combination with two organic substances, potassium humate and chitosan in controlling Meloidogyne incognita on tomato. All treatments caused greater decreases in parameters of M. incognita in comparison to the control treatment (nematode only) and this led to noticeable enhancements in growth and yield of tomato. The lowest numbers of eggmasses, eggs/eggmass, galls/root, females/root, and second stage juveniles/250?g soil were recorded due to the combination of T. harzianum (10¹⁰ spore/ml) with chitosan and potassium humate after 120 days from the transplanting of tomato seedlings. Also, this treatment showed the best promotion for all tomato parameters (lengths and weights of shoots and roots, and productivity). So, mixing chitosan, potassium and T. harzianum is highly recommended to be used as an effective bio-nematicide against M. incognita on tomato plants.     

Isolation of nematophagous fungi from eggs and females of Meloidogyne spp. and evaluation of their biological control potential

Fungi were isolated from Meloidogyne spp. eggs and females on 102 field-collected root samples in China. Of the 235 fungi isolated (representing 18 genera and 26 species), the predominant fungi were Fusarium spp. (42.1% of the isolates collected), Fusarium oxysporum (13.2%), Paecilomyces lilacinus (12.8%), and Pochonia chlamydosporia (8.5%). The isolates were screened for their ability to parasitise Meloidogyne incognita eggs in 24-well tissue culture plates in two different tests. The percentage of eggs parasitised by the fungi, the numbers of unhatched eggs and alive and dead juveniles were counted at 4 and 7 days after inoculation. The most promising fungi included five Paecilomyces isolates, 10 Fusarium isolates, 10 Pochonia isolates and one Acremonium isolate in test 1 or test 2. Paecilomyces lilacinus YES-2 and P. chlamydosporia HDZ-9 selected from the in vitro tests were formulated in alginate pellets and evaluated for M. incognita control on tomato in a greenhouse by adding them into a soil with sand mixture at rates of 0.2, 0.4, 0.8 and 1.6% (w/w). P. lilacinus pellets at the highest rate (1.6%) reduced root galling by 66.7%. P. chlamydosporia pellets at the highest rate reduced the final nematode density by 90%. The results indicate that P. lilacinus and P. chlamydosporia as pellet formulation can effectively control root-knot nematodes.     

Nematicidal screening of essential oils and potent toxicity of Dysphania ambrosioides essential oil against Meloidogyne incognita in vitro and in vivo

It is known that some plant essential oils have pesticide activities. Among the 29 oils evaluated in this study, 14 showed nematicidal activities of 8 to 100% at the concentration of 1,000 μg/ml, compared with a control of 0.01 g/ml Tween 80^®. At a lower concentration of 500 μg/ml, only Dysphania ambrosioides oil caused >90% mortality of second‐stage juveniles (J2) of Meloidogyne incognita. The LC₅₀ and LC₉₅ values for D. ambrosioides oil were 307 μg/ml and 580 μg/ml, respectively. M. incognita eggs placed in D. ambrosioides oil solutions had a significant reduction in J2 hatching compared with controls. Therefore, the oil had a toxic effect on both eggs and J2 of M. incognita. This was in contrast to nematicides on the market that act efficiently only on J2. When J2 were placed in D. ambrosioides oil at its LC₅₀ concentration and inoculated onto tomato plants, the reduction in numbers of galls and eggs was 99.5% and 100%, respectively. Dysphania ambrosioides oil applied to the potting substrate of plants at a concentration of 1,100 μg/ml significantly reduced the number of galls and eggs of M. incognita, whereas a concentration of 800 μg/ml only reduced the number of eggs compared with the controls (Tween 80^® and water). The main components of the D. ambrosioides oil detected by gas chromatography–mass spectrometry were (Z)‐ascaridole (87.28%), E‐ascaridole (8.45%) and p‐cymene (3.35%), representing 99.08% of the total oil composition. Given its nematicidal activity, D. ambrosioides oil represents an exciting raw material in the search for new bioactive molecules for the pesticide industry.     

Activity of four nematicides against Meloidogyne incognita race 2 on tomato plants

Meloidogyne incognita is one of the most important causes of disease in protected vegetable cultivation in North China Plain, but chemical control options for it are currently limited. In the present study, we measured activity of four nematicides with M. incognita race 2 in the laboratory and greenhouse pots. Fluensulfone and fluopyram had a greater negative effect on the motility of M. incognita second-stage juveniles (J2) than avermectin B1a (AV-B1a) and fosthiazate had, especially at low concentration, with respective EC₅₀ values of 0.29, 0.13, 0.68 and 2.48 mg/L. AV-B1a significantly and uniquely inhibited egg hatching, which indicated that it was the only nematicide that could readily be transported across the eggshell. In greenhouse pots, fluensulfone (10 mg⁻¹) and fluopyram (1 and 10 mg⁻¹) caused the greatest inhibition of formation of galls in tomato roots, with decreases of 98.6%, 96.2% and 99.2%, respectively. This indicated that some M. incognita J2 lost their root penetration ability without losing their motility.     

Efficacy of new nematicides for managing Meloidogyne incognita in tomato crop

New non‐fumigant nematicides (fluensulfone, fluopyram and fluazaindolizine) were tested in greenhouse tomato trials aiming to evaluate its efficacy on the control of Meloidogyne incognita soil and root populations and plant produtivity. Plants of the cultivar Red Gnome were transplanted into 2,500 cm³ fibre pots inoculated with 200 eggs of M. incognita/100 cm³ of soil and treated with fluensulfone, fluopyram and fluazaindolizine, in two rates each. After eight weeks of incubation the plants were evaluated for fresh root and shoot weight, weight and number of fruits, egg mass number, population density and nematode reproduction factor. All nematicide treatments reduced the root gall index, the number of M. incognita egg masses, eggs/g root and the nematode reproduction factor when compared to the non‐treated control.     

Phythonematoxic properties and nematicidal potential of Tithonia diversifolia extract and residue on Meloidogyne incognita infecting yam (Discoria rotundata)

A laboratory study and a screen house experiment were conducted to determine the phythonematoxic properties and nematicidal potential of extract and residue of Tithonia diversifolia (Hemsl.) A. Gray on Meloidogyne incognita, (Kofoid and White) Chitwood infecting yam. Alkaloids and saponins were found to be present in the constituents of Tithonia ethanol extract. Also, Tithonia water extract significantly (P ≤ 0.05) inhibits M. incognita egg hatch by 98% from 2 days after incubation (DAI) and was more evidenced with 100% inhibition at 9 DAI in the in vitro studies. M. incognita (5000 eggs/plant) reproduction, number of eggs and juveniles and galling were significantly (P ≤ 0.05) suppressed by Tithonia residue treatment at a rate of 30 tons/ha on yam (Discoria rotundata) in the screen house. The extract and residue of T. diversifolia have potential for the control of root-knot nematodes.     

Nematicidal effect of an Amaranthus hypochondriacus L. cystatin (AhCPI) on the root-knot nematode Meloidogyne incognita (Kofoid and White) Chitwood

The root-knot nematode Meloidogyne incognita is one of the most damaging plant parasitic nematodes in the world. In this study, the effect of cystatin from Amaranthus hypochondriacus (AhCPI) as a potential control agent for M. incognita was explored. In vitro bioassays demonstrated that AhCPI affects the growth and development of eggs and the infectivity of juveniles (J2) of M. incognita, such as mortality and slower development, showing characteristic tissue damage. Mortality levels were quantified by Probit analysis, estimating LC₅₀s of 1.4 mg/mL for eggs and 0.028 mg/mL for J2. In planta bioassays showed that infected tomato seedlings treated with 0.056 mg/mL of AhCPI showed a 60% reduction in the number of galls, as compared with untreated J2-inoculated seedlings. Under greenhouse conditions, three applications of 10 mL of AhCPI (1.4 mg/mL) in the soil around the stem of M. incognita-infected tomato plants, reduced the number of galls by 93 ± 8%, as compared to the control M. incognita-infected plants. The application of AhCPI to the infected plants increased the yield (10.7%) of harvested tomato fruits, as compared to infected plants. These results show the potential of AhCPI for the control of M. incognita in tomato plants.     

A new oospecies of Similifaveoloolithidae from the Xiuning Basin,Late Cretaceous of Anhui,China

Well-preserved dinosaur eggs from the Cretaceous Huizhou Formation in the Xiuning Basin, Anhui Province, China, are analysed in this paper. We describe a new oospecies, Similifaveoloolithus qiyunshanensis, based on several distinct characters of external morphology, size, eggshell thickness, and internal microstructure. Radial sections of this new oospecies show branched eggshell units with a fused layer near the outer surface, while numerous irregular pores and cones constitute a honeycomb pattern in tangential sections. The discovery of S. qiyunshanensis expands the distribution of Similifaveoloolithidae dinosaur eggs in China and provides new fossils for researching dinosaur eggshell formation mechanisms which are different from those of the currently known oofamilies. The dinosaur-egg-bearing strata in the Huizhou Formation have been dated to the early Late Cretaceous (Cenomanian-Turonian) on the basis of a similar dinosaur egg assemblage in the Tiantai Basin in Zhejiang Province. The eggs described in this paper are thought to have been laid in a buried nest while enrichment of trace elements in eggshells may have been caused by their ingestion into the body of the dinosaur producer. We suggest that the paleoclimate of this habitat was semi-arid to arid and that this environment was favourable for the preservation of eggs.http://zoobank.org/LSIDurn:lsid:zoobank.org:pub:9D63A51F-5A4B-4249-901D-42A33E9FA933     

Characterisation of Isaria fumosorosea isolates and their virulence toward the Diamondback Moth,Plutella xylostella

Some morphological and physiological characteristics of an Isaria fumosorosea isolate with diminished virulence, IFCF01-D, and its parent isolate, IFCF01, were evaluated and laboratory bioassays were performed to assess their virulence against Plutella xylostella. The relationship among these traits and virulence against P. xylostella is discussed. There were no significant differences in conidial viability, spore production and the time required for 50% germination (GT₅₀). Spore viability after incubation for 24 h at 25°C was greater than 98% for both isolates tested. Spore production on potato dextrose agar after 14 days incubation at 25°C was 4.68 × 10⁸ and 4.59 × 10⁸ conidia/mL for IFCF01 and IFCF01-D, respectively. When exposed to high temperatures (40, 45, 50 or 55°C) through a water bath for 10 min, conidial germination ranged from 0.83% to 84.0% for IFCF01 and 0% to 86% for IFCF01-D. Germination rate showed a negative relationship with the exposure temperature for both isolates. The per cent germination of isolate IFCF01 24 h after ultraviolet (UV) radiation (18 W, 240–260 nm) varied from 0% to 92% and 0% to 81% for IFCF01-D. Germination rate and the exposure time exhibited a negative correlation for both isolates tested. Conidial surface hydrophobicity of IFCF01 (60%) was significantly higher than that of isolate IFCF01-D (53%). Subsequently, using the cicada exuviae as the substrate for enzymatic analysis, Pr1 and chitinase activity demonstrated the contrasting virulence traits: higher specific activities for the more virulent IFCF01 and lower enzymatic levels for isolate IFCF01-D.     

Hosts’ Responses to Parasitic Eggs: Which Cues Elicit Hosts’ Egg Discrimination?

Many hosts of the common cuckoo (Cuculus canorus) exhibit egg recognition, and reject parasitic eggs. How do hosts discriminate cuckoo eggs from their own? Hosts might be able to recognize their own eggs using the specific pigment pattern on the outer eggshell surface, which may serve as a cue for recognition. We tested if patterns of egg pigments (spottedness) contain this information by manipulating spot density of great reed warbler eggs (Acrocephalus arundinaceus). We also manipulated the colour of eggs when the original spot pattern remained the same. Spot density (approximately 15–75%) did not significantly affect rejection rate (8–20% rejection), but when spots fully covered the eggs, i.e. the eggshell was plain dark brown, rejection rate increased abruptly to 100%. A loglinear model revealed the significant influence of colour on rejection rates, although there was no interactive effect between spottedness and colour. Our results strongly support the differential use of egg markers in host’s egg discrimination, suggesting that spot density has limited importance compared to eggshell colour.     

Biocontrol activity of salt tolerant Streptomyces isolates against phytopathogens causing root rot of sugar beet

Biological control of fungi causing root rot on sugar beet by native Streptomyces isolates (C and S2) was evaluated in this study. The dry weight and colony forming unit (CFU) of S2 and C increased when 300 mM NaCl was added to medium. The in vitro antagonism assays showed that both isolates had inhibitory effect against Rhizoctonia solani AG-2, Fusarium solani and Phytophthora drechsleri. In dual culture, Streptomyces isolate C inhibited mycelial growth of R. solani, F. solani and P. drechsleri 45%, 53% and 26%, respectively. NaCl treatment of medium increased biocontrol activity of soluble and volatile compounds of isolate C and S2. After salt treatment, growth inhibition of R. solani, F. solani and P. drechsleri by isolate C increased up to 59%, 70% and 79%, respectively. To elucidate the mode of antagonism, protease, chitinase, beta glucanase, cellulase, lipase and α-amylase activity and siderophore and salicylic acid (SA) production were evaluated. Both isolates showed protease, chitinase and α-amylase activity. Also, biosynthesis of siderophore was detectable for both isolates. Production of siderophore and activity of protease and α-amylase increased after adding salt for both isolates. In contrast, chitinase activity decreased significantly. Production of SA, beta glucanase and lipase by isolate S2 and biosynthesis of cellulase by isolate C were observed in presence and absence of NaCl. Soil treatment with Streptomyces isolate C inhibited root rot of sugar beet caused by P. drechsleri, R. solani and F. solani. Results of this study showed that these two Streptomyces isolates had potential to be utilized as biocontrol agent against fungal diseases especially in saline soils.