Biological control of the native endophytic fungus Pochonia chlamydosporia from the root nodule of Dolichos lablab on Fusarium wilt of banana TR4.

Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense, Tropical Race 4 (TR4) is a soil-borne disease, and it is devastating. At present, the biological control using antagonistic microorganisms to mitigate TR4 is one of the best strategies as a safe and green way. Yunnan has abundant and diverse microbial resources. Using the dual-culture method, the antagonistic endophytic fungi against TR4 were isolated and screened from the root nodule of Dolichos lablab. The effect of the highest antagonistic activity strain on the morphology of the TR4 mycelium was observed using the scanning electron microscope. According to morphological characteristics and sequence analysis, the strain was identified. The biocontrol effect and plant growth promotion were investigated by greenhouse pot experiment. Using the confocal laser scanning microscope and the real-time fluorescence quantitative PCR, the dynamics of TR4 infestation and the TR4 content in banana plant roots and corms would also be detected. In this study, 18 native endophytic fungi were isolated from a root nodule sample of Dolichos lablab in the mulch for banana fields in Yuxi, Yunnan Province, China. The YNF2217 strain showed a high antagonistic activity against TR4 in plate confrontation experiments, and the inhibition rate of YNF2217 is 77.63%. After TR4 culture with YNF2217 for 7 days in plate confrontation experiments, the morphology of the TR4 mycelium appeared deformed and swollen when observed under a scanning electron microscope. According to morphological characteristics and sequence analysis, the strain YNF2217 was identified as Pochonia chlamydosporia. In the greenhouse pot experiment, the biocontrol effect of YNF2217 fermentation solution on TR4 was 70.97% and 96.87% on banana plant leaves and corms, respectively. Furthermore, YNF2217 significantly promoted the growth of banana plants, such as plant height, leaf length, leaf width, leaf number, pseudostem girth, and both the aboveground and underground fresh weight. Observations of TR4 infestation dynamics in banana roots and corms, along with real-time fluorescence quantitative PCR, verified that YNF2217 inoculation could significantly reduce the TR4 content. Therefore, YNF2217 as P. chlamydosporia, which was found first time in China and reported here, is expected to be an important new fungal resource for the green control of Fusarium wilt of banana in the future.

Nematicidal glycosylated resorcylic acid lactones from the fungus Pochonia chlamydosporia PC-170 and their key biosynthetic genes.

Chemical study of the nematicidal biocontrol fungus Pochonia chlamydosporia PC-170 led to discovery of six resorcylic acid lactones (RALs), including three nematicidal glycosylated RALs, monocillin VI glycoside (1), colletogloeolactone A (2) and monocillin II glycoside (3), and three antibacterial non-glycosylated RALs, monocillin VI (4), monocillin IV (5) and monocillin II (6). The planar structure of the new compound monocillin VI glycoside (1) was elucidated using HRESIMS and NMR data, and its monosaccharide configuration was further determined through sugar hydrolysis experiment and GC-MS analysis method. Furthermore, their two biosynthetic-related PKS genes, pchE and pchI, were identified through the gene knockout experiment. The glycosylated RALs 1-3 exhibited nematicidal activity against Meloidogyne incognita, with LC50 values of 94, 152 and 64 µg/mL, respectively, and thus had great potential in the development of new nematicidal natural products to control M. incognita in the future.

Chitosan from Marine Amphipods Inhibits the Wilt Banana Pathogen Fusarium oxysporum f. sp. Cubense Tropical Race 4.

In this work, we extracted chitosan from marine amphipods associated with aquaculture facilities and tested its use in crop protection. The obtained chitosan was 2.5 ± 0.3% of initial ground amphipod dry weight. The chemical nature of chitosan from amphipod extracts was confirmed via Raman scattering spectroscopy and Fourier transform infrared spectroscopy (FTIR). This chitosan showed an 85.7-84.3% deacetylation degree. Chitosan from biofouling amphipods at 1 mg·mL-1 virtually arrested conidia germination (ca. sixfold reduction from controls) of the banana wilt pathogenic fungus Fusarium oxysporum f. sp cubense Tropical Race 4 (FocTR4). This concentration reduced (ca. twofold) the conidia germination of the biocontrol fungus Pochonia chlamydosporia (Pc123). Chitosan from amphipods at low concentrations (0.01 mg·mL-1) still reduced FocTR4 germination but did not affect Pc123. This is the first time that chitosan is obtained from biofouling amphipods. This new chitosan valorizes aquaculture residues and has potential for biomanaging the diseases of food security crops such as bananas.

Polyketides from the fungus Pochonia chlamydosporia and their bioactivities.

Three previously undescribed griseofulvin derivatives, namely pochonichlamydins A-C, one small polyketide, namely pochonichlamydin D, together with nine known compounds, have been isolated from cultures of the fungus Pochonia chlamydosporia. Their structures with absolute configurations were elucidated on the basis of extensive spectrometric methods and single-crystal X-ray diffraction. Dechlorogriseofulvin and griseofulvin exhibited inhibitory activities against Candida albicans at the concentration of 100 µM, with inhibition rates of 69.1% and 56.3%, respectively. Meanwhile, pochonichlamydin C showed mild cytotoxicity against the human cancer MCF-7 cell line with an IC50 value of 33.1 µM.

Chitosan and nematophagous fungi for sustainable management of nematode pests.

Plants are exposed to large number of threats caused by herbivores and pathogens which cause important losses on crops. Plant pathogens such as nematodes can cause severe damage and losses in food security crops worldwide. Chemical pesticides were extendedly used for nematode management. However, due to their adverse effects on human health and the environment, they are now facing strong limitations by regulatory organisations such as EFSA (European Food Safety Authority). Therefore, there is an urgent need for alternative and efficient control measures, such as biological control agents or bio-based plant protection compounds. In this scenario, chitosan, a non-toxic polymer obtained from seafood waste mainly, is becoming increasingly important. Chitosan is the N-deacetylated form of chitin. Chitosan is effective in the control of plant pests and diseases. It also induces plants defence mechanisms. Chitosan is also compatible with some biocontrol microorganisms mainly entomopathogenic and nematophagous fungi. Some of them are antagonists of nematode pests of plants and animals. The nematophagous biocontrol fungus Pochonia chlamydosporia has been widely studied for sustainable management of nematodes affecting economically important crops and for its capability to grow with chitosan as only nutrient source. This fungus infects nematode eggs using hyphal tips and appressoria. Pochonia chlamydosporia also colonizes plant roots endophytically, stimulating plant defences by induction of salicylic and jasmonic acid biosynthesis and favours plant growth and development. Therefore, the combined use of chitosan and nematophagous fungi could be a novel strategy for the biological control of nematodes and other root pathogens of food security crops.

Pochonia chlamydosporia Isolate PC-170-Induced Expression of Marker Genes for Defense Pathways in Tomatoes Challenged by Different Pathogens.

Pochonia chlamydosporia is a fungal parasite of nematode eggs. Studies have shown that some strains of Pochonia chlamydosporia can promote plant growth and induce plants' systemic resistance to root-knot nematodes by colonizing in their roots. This study aimed to verify the effect of the PC-170 strain on tomato growth and systemic resistance. Split-root experiments were conducted to observe the systemic resistance induced by PC-170. To explore the defense pathway that was excited due to the colonization by PC-170, we tested the expression of marker genes for defense pathways, and used mutant lines to verify the role of plant defense pathways. Our results showed that PC-170 can colonize roots, and promotes growth. We found a role for jasmonic acid (JA) in modulating tomato colonization by PC-170. PC-170 can activate tomato defense responses to reduce susceptibility to infection by the root-knot nematode Meloidogyne incognita, and induced resistance to some pathogens in tomatoes. The marker genes of the defense pathway were significantly induced after PC-170 colonization. However, salicylic acid (SA)- and jasmonic acid (JA)-dependent defenses in roots were variable with the invasion of different pathogens. Defense pathways play different roles at different points in time. SA- and JA-dependent defense pathways were shown to cross-communicate. Different phytohormones have been involved in tomato plants' responses against different pathogens. Our study confirmed that adaptive JA signaling is necessary to regulate PC-170 colonization and induce systemic resistance in tomatoes.

Putative LysM Effectors Contribute to Fungal Lifestyle.

Fungal LysM effector proteins can dampen plant host-defence responses, protecting hyphae from plant chitinases, but little is known on these effectors from nonpathogenic fungal endophytes. We found four putative LysM effectors in the genome of the endophytic nematophagous fungus Pochonia chlamydosporia (Pc123). All four genes encoding putative LysM effectors are expressed constitutively by the fungus. Additionally, the gene encoding Lys1-the smallest one-is the most expressed in banana roots colonised by the fungus. Pc123 Lys1, 2 and 4 display high homology with those of other strains of the fungus and phylogenetically close entomopathogenic fungi. However, Pc123 Lys3 displays low homology with other fungi, but some similarities are found in saprophytes. This suggests evolutionary divergence in Pc123 LysM effectors. Additionally, molecular docking shows that the NAcGl binding sites of Pc123 Lys 2, 3 and 4 are adjacent to an alpha helix. Putative LysM effectors from fungal endophytes, such as Pc123, differ from those of plant pathogenic fungi. LysM motifs from endophytic fungi show clear conservation of cysteines in Positions 13, 51 and 63, unlike those of plant pathogens. LysM effectors could therefore be associated with the lifestyle of a fungus and give us a clue of how organisms could behave in different environments.

Metarhizium: jack of all trades, master of many.

The genus Metarhizium and Pochonia chlamydosporia comprise a monophyletic clade of highly abundant globally distributed fungi that can transition between long-term beneficial associations with plants to transitory pathogenic associations with frequently encountered protozoans, nematodes or insects. Some very common 'specialist generalist' species are adapted to particular soil and plant ecologies, but can overpower a wide spectrum of insects with numerous enzymes and toxins that result from extensive gene duplications made possible by loss of meiosis and associated genome defence mechanisms. These species use parasexuality instead of sex to combine beneficial mutations from separate clonal individuals into one genome (Vicar of Bray dynamics). More weakly endophytic species which kill a narrow range of insects retain sexuality to facilitate host-pathogen coevolution (Red Queen dynamics). Metarhizium species can fit into numerous environments because they are very flexible at the genetic, physiological and ecological levels, providing tractable models to address how new mechanisms for econutritional heterogeneity, host switching and virulence are acquired and relate to diverse sexual life histories and speciation. Many new molecules and functions have been discovered that underpin Metarhizium associations, and have furthered our understanding of the crucial ecology of these fungi in multiple habitats.

Ketamine can be produced by Pochonia chlamydosporia: an old molecule and a new anthelmintic?

BACKGROUND: Infection by nematodes is a problem for human health, livestock, and agriculture, as it causes deficits in host health, increases production costs, and incurs a reduced food supply. The control of these parasites is usually done using anthelmintics, which, in most cases, have not been fully effective. Therefore, the search for new molecules with anthelmintic potential is necessary. METHODS: In the present study, we isolated and characterized molecules from the nematophagous fungus Pochonia chlamydosporia and tested these compounds on three nematodes: Caenorhabditis elegans; Ancylostoma ceylanicum; and Ascaris suum. RESULTS: The ethyl acetate extract showed nematicidal activity on the nematode model C. elegans. We identified the major substance present in two sub-fractions of this extract as ketamine. Then, we tested this compound on C. elegans and the parasites A. ceylanicum and A. suum using hamsters and mice as hosts, respectively. We did not find a difference between the animal groups when considering the number of worms recovered from the intestines of animals treated with ketamine (6 mg) and albendazole (P > 0.05). The parasite burden of larvae recovered from the lungs of mice treated with ketamine was similar to those treated with ivermectin. CONCLUSIONS: The results presented here demonstrate the nematicidal activity of ketamine in vitro and in vivo, thus confirming the nematicidal potential of the molecule present in the fungus P. chlamydosporia may consist of a new method of controlling parasites.

In vitro biological control of bovine parasitic nematodes by Arthrobotrys cladodes, Duddingtonia flagrans and Pochonia chlamydosporia under different temperature conditions.

Variations in temperature can affect the development of nematophagous fungi, especially when they are used in the biological control of parasitic nematodes in the pastures where cattle are reared. The aim of this work was to evaluate the effects of temperature on the performance of nematophagous fungi in the biological control of bovine parasitic nematodes. The mycelial growth, chlamydospore production and nematicidal activity of Duddingtonia flagrans, Arthrobotrys cladodes and Pochonia chlamydosporia were evaluated at 15, 20, 25, 30 and 35°C. The fungal strains achieved mycelial growth, chlamydospore production and nematicidal activity on parasitic nematodes under all temperature conditions tested. The fungi showed higher growth at intermediate temperatures (20, 25 and 30°C) than at the extremes of 15 and 35°C. At 25 and 30°C, D. flagrans realized 96.8 and 94.5% nematicidal activity on bovine parasitic nematodes, respectively. Arthrobotrys cladodes effected nematicidal activity of 85.3 and 83.5%, at 20 and 25°C, respectively. At 20 and 30°C, P. chlamydosporia achieved nematicidal activity of 81.3 and 87.4%, respectively. The maximum chlamydospore production was reached at 20, 25 and 30°C for D. flagrans, at 20 and 25°C for A. cladodes and P. chlamydosporia. The results of this study demonstrated that the tested fungal strains of D. flagrans, A. cladodes and P. chlamydosporia, when used in the biological control of bovine parasitic nematodes, were not limited by in vitro temperature variations. Therefore, the use of these strains of fungi as biological control agents of parasitic nematodes is promising.

Arthrobotrys cladodes and Pochonia chlamydosporia: Nematicidal effects of single and combined use after passage through cattle gastrointestinal tract.

Nematodiosis are responsible for financial losses in cattle production systems due to treatment costs, falling production rates and animal deaths. The incorporation of sodium alginate pellets containing nematophagous fungi in the bovine diet is a method for the control of nematodiosis. The aims of this study were to evaluate the viability of Arthrobotrys cladodes and Pochonia chlamydosporia contained in sodium alginate pellets after passage through the bovine gastrointestinal tract, as well as to evaluate the effects of single and combined use of these fungi against bovine parasitic nematodes. The reduction in parasitic nematode infective larvae of bovines by the combined use of Arthrobotrys cladodes and Pochonia chlamydosporia was more than the reduction in infective larvae by Arthrobotrys cladodes or Pochonia chlamydosporia alone in the collections 12, 24 and 36 h after giving the pellets to the animals. Arthrobotrys cladodes and Pochonia chlamydosporia combined promoted maximum reduction of infective larvae of 86.3%. Therefore, the combination of Arthrobotrys cladodes and Pochonia chlamydosporia may be an effective method to control gastrointestinal nematodiosis affecting grazing cattle.

Arthrobotrys cladodes and Pochonia chlamydosporia in the biological control of nematodiosis in extensive bovine production system.

Cattle production in extensive systems favours the occurrence of gastrointestinal nematodes, and the use of nematophagous fungi complements the control strategies for these nematodes. The aim of this study was to evaluate the effectiveness of the fungi Arthrobotrys cladodes and Pochonia chlamydosporia in the biological control of gastrointestinal parasitic nematodes in grazing cattle. Twenty-four calves were randomly divided into four groups and allocated to independent paddocks from February 2018 to January 2019. In the first group, the animals received pellets containing P. chlamydosporia. In the second group, the animals received pellets containing A. cladodes. In the third group, the animals received pellets containing a combination of the fungi A. cladodes and P. chlamydosporia. In the control group, the animals received pellets without fungus. The combined use of A. cladodes and P. chlamydosporia showed greater efficacy in the biological control of bovine gastrointestinal parasitic nematodes than the same fungi used separately. The parasite load was lower and weight gain was greater (P ⩽ 0.05) in the groups of cattle treated with nematophagous fungi. Therefore, the use of A. cladodes and P. chlamydosporia is promising in the biological control of nematodiosis in cattle.

Time-dependent effects of Pochonia chlamydosporia endophytism on gene expression profiles of colonized tomato roots.

A transcriptome analysis was produced from tomato roots inoculated with the hyphomycete Pochonia chlamydosporia at three different times. Gene expression data were also yielded from fungus grown in vitro or endophytic. A next-generation sequencing (NGS) and network analysis approach were applied. We identified 3.676 differentially expressed tomato genes (DEG), highlighting a core of 93 transcripts commonly down- or upregulated at every time point, shedding light on endophytism process. Functional categories related to plant information-processing system, which recognizes, percepts, and transmits signals, were associated with gene upregulated early in time, with higher representations in processes such as plant defense regulation later in time. Network analysis of a DEG subset showed dominance of MAP kinase hubs in the uninoculated control samples, replaced by an increased centrality of WRKY transcription factor and ETR-ethylene response factor genes in the colonized roots. Fungus genes expressed during progression of plant colonization, therefore related to the host colonization process or endophytism persistence, were also identified. Data provided a high-resolution insight on tomato transcriptome changes as induced by endophytism, highlighting a specific modulation of stress-responsive transcripts, related to a selective activation of defense pathways, likely required by the fungus to establish a persistent endophytic lifestyle.

Commercial Formulates of Trichoderma Induce Systemic Plant Resistance to Meloidogyne incognita in Tomato and the Effect Is Additive to That of the Mi-1.2 Resistance Gene.

Meloidogyne is the most damaging plant parasitic nematode genus affecting vegetable crops worldwide. The induction of plant defense mechanisms against Meloidogyne in tomato by some Trichoderma spp. strains has been proven in pot experiments, but there is no information for tomato bearing the Mi-1.2 resistance gene or for other important fruiting vegetable crops. Moreover, Trichoderma is mostly applied for managing fungal plant pathogens, but there is little information on its effect on nematode-antagonistic fungi naturally occurring in soils. Thus, several experiments were conducted to determine (i) the ability of two commercial formulates of Trichoderma asperellum (T34) and Trichoderma harzianum (T22) to induce systemic resistance in tomato and cucumber against an avirulent Meloidogyne incognita population in split-root experiments; (ii) the effect of combining T34 with tomato carrying the Mi-1.2 resistance gene to an avirulent M. incognita population in sterilized soil; and (iii) the effect of combining T34 with tomato carrying the Mi-1.2 resistance gene to a virulent M. incognita population in two suppressive soils in which Pochonia chlamydosporia is naturally present, and the effect of T34 on the level of P. chlamydosporia egg parasitism. Both Trichoderma formulates induced resistance to M. incognita in tomato but not in cucumber. In tomato, the number of egg masses and eggs per plant were reduced by 71 and 54% by T34, respectively. T22 reduced 48% of the number of eggs per plant but not the number of egg masses. T34 reduced the number of eggs per plant of the virulent M. incognita population in both resistant and susceptible tomato cultivars irrespective of the suppressive soil, and its effect was additive with the Mi-1.2 resistance gene. The percentage of fungal egg parasitism by P. chlamydosporia was not affected by the isolate T34 of T. asperellum.

Pochoniolides A and B, new antioxidants from the fungal strain Pochonia chlamydosporia var. spinulospora FKI-7537.

New natural products, designated pochoniolides A and B, were isolated from the cultured broth of fungal strain FKI-7537 using a physicochemical screening methodology. Strain FKI-7537 was isolated from a soil sample collected at Niijima, Tokyo, Japan and identified as Pochonia chlamydosporia var. spinulospora by morphological characteristics and DNA sequence analysis. The chemical structures of pochoniolides A and B were elucidated by NMR and mass spectra and found to be new compounds consisting of a muconolactone moiety connected with a chromone unit. Pochoniolides A and B were identified as racemate mixtures using data on optical rotation and circular dichroism spectra. Furthermore, enantiomers of pochoniolide B, pochoniolides B1 and B2, were separated using a chiral HPLC column. Pochoniolides A and B showed hydroxyl radical-scavenging and singlet oxygen-quenching activities.

Statistical tools application on dextranase production from Pochonia chlamydosporia (VC4) and its application on dextran removal from sugarcane juice

ABSTRACT The aim of this study was to optimize the dextranase production by fungus Pochonia chlamydosporia (VC4) and evaluate its activity in dextran reduction in sugarcane juice. The effects, over the P. chlamydosporia dextranase production, of different components from the culture medium were analyzed by Plackett-Burman design and central composite design. The response surface was utilized to determine the levels that, among the variables that influence dextranase production, provide higher production of these enzymes. The enzymatic effect on the removal of dextran present in sugarcane juice was also evaluated. It was observed that only NaNO3 and pH showed significant effect (p<0.05) over dextranase production and was determined that the levels which provided higher enzyme production were, respectively, 5 g/L and 5.5. The dextranases produced by fungus P. chlamydosporia reduced by 75% the dextran content of the sugarcane juice once treated for 12 hours, when compared to the control treatment.

Evaluation of Pochonia chlamydosporia and Purpureocillium lilacinum for Suppression of Meloidogyne enterolobii on Tomato and Banana.

Meloidogyne enterolobii is one of the most important root-knot nematode in tropical regions, due to its ability to overcome resistance mechanisms of a number of host plants. The lack of new and safe active ingredients against this nematode has restricted control alternatives for growers. Egg-parasitic fungi have been considered as potential candidates for the development of bionematicides. In tissue culture plates, Pochonia chlamydosporia (var. catenulata and chlamydosporia) and Purpureocillium lilacinum strains were screened for their ability to infect eggs of the root-knot nematode M. enterolobii on water-agar surfaces. Reduction in the hatching of J2 varied from 13% to 84%, depending on strain. The more efficacious strains reduced hatchability of J2 by 57% to 84% when compared to untreated eggs, but average reductions were only 37% to 55% when the same strains were applied to egg masses. Combinations of fungal isolates (one of each species) did not increase the control efficacy in vitro. In experiments in which 10,000 nematode eggs were inoculated per plant, reductions in the number of eggs after 12 months were seen in three of four treatments in banana plants, reaching 34% for P. chlamydosporia var. catenulata. No significant reductions were seen in tomato plants after 3 mon. In another experiment with tomato plants using either P. chlamydosporia var. catenulata or P. lilacinum, the number of eggs was reduced by 34% and 44%, respectively, when initial infestation level was low (500 nematode eggs per plant), but tested strains were not effective under a moderate infestation level (5,000 eggs per plant). Under all infestation levels tested in this work, gall and egg mass indexes (MI) did not differ from the untreated controls, bringing concerns related to the practical adoption of this control strategy by farmers. In our opinion, if the fungi P. chlamydosporia and P. lilacinum are to be used as biocontrol tools toward M. entorolobii, they should focus on agricultural settings with low soil infestation levels and within an IPM approach.

Arabidopsis thaliana root colonization by the nematophagous fungus Pochonia chlamydosporia is modulated by jasmonate signaling and leads to accelerated flowering and improved yield.

Pochonia chlamydosporia has been intensively studied in nematode control of different crops. We have investigated the interaction between P. chlamydosporia and the model system Arabidopsis thaliana under laboratory conditions in the absence of nematodes. This study demonstrates that P. chlamydosporia colonizes A. thaliana. Root colonization monitored with green fluorescent protein-tagged P. chlamydosporia and quantitative PCR (qPCR) quantitation methods revealed root cell invasion. Fungal inoculation reduced flowering time and stimulated plant growth, as determined by total FW increase, faster development of inflorescences and siliques, and a higher yield in terms of seed production per plant. Precocious flowering was associated with significant expression changes in key flowering-time genes. In addition, we also provided molecular and genetic evidence that point towards jasmonate signaling as an important factor to modulate progression of plant colonization by the fungus. Our results indicate that P. chlamydosporia provides benefits to the plant in addition to its nematophagous activity. This report highlights the potential of P. chlamydosporia to improve yield in economically important crops.

Biocontrol agents in the management of Meloidogyne incognita in tomato / Agentes de biocontrole no manejo de Meloidogyne incognita em tomateiro

ABSTRACT: Recognized as prominent among the principal pests of tomato crop, the root-knot nematode (Meloidogyne incognita) has generated much study with regards to control methods. This study aimed to assess the efficiency of biological products in decreasing the M. incognita population in tomato. Four experiments were conducted under greenhouse conditions. Two of these experiments had the purpose to determine the efficiency of NemOut™ (Bacillus subtilis + B. licheniformis + Trichoderma longibrachiatum), and the other two to assess the efficiency of other different biological products in M. incognita management. Tomato plants of the cultivar Santa Cruz Kada Giant were inoculated using 2000 eggs and J2 of M. incognita. In the first two experiments doses corresponding to 0, 4, 6, 8 and 10kg ha-1 of NemOut™ were applied after inoculation. In the second study, the treatments consisted of the use of biological products containing different other biocontrol agents. Assessments were made at 45 and 65 days after inoculation (DAI) to evaluate plant height, fresh shoot and fresh root weigh, gall index and egg mass index, population density and reproduction factor (RF). The NemOut™ doses reduced the M. incognita population density and RF at 45 DAI. The agent Pochonia chlamydosporia showed greater efficiency in controlling M. incognita on tomato. Biological products and the different doses of NemOut™ had no influence on the tomato plant development.

RESUMO: O nematoide das galhas (Meloidogyne incognita) é relatado como uma das principais pragas da cultura do tomate, sendo importante o estudo de alternativas para o seu manejo. O presente estudo objetivou avaliar a eficiência de produtos biológicos na redução da população de M. incognita em tomate. Foram conduzidos quatro experimentos em casa de vegetação, dois visando avaliar a eficiência de doses de NemOut™ (Bacillus subtilis + B. licheniformis + Trichoderma longibrachiatum), e outros dois para avaliar a eficiência de diferentes produtos biológicos no controle de M. incognita. Plantas de tomate Santa Cruz Kada Gigante foram inoculadas com 2000 ovos e J2 de M. incognita. No primeiro estudo foram utilizadas doses equivalentes a 0, 4, 6, 8 e 10kg ha-1 de NemOut™, aplicadas após a inoculação. No segundo estudo os tratamentos constituíram de produtos biológicos com diferentes agentes de biocontrole. As avaliações foram realizadas 45 e 65 dias após a inoculação (DAI), determinando-se altura de plantas, massa fresca da parte aérea e da raiz, índice de galhas e de massas de ovos, densidade populacional e fator de reprodução (FR). Doses crescentes de NemOut™ reduziram a densidade populacional e o FR de M. incognita, aos 45 DAI. O agente Pochonia chlamydosporia mostrou-se mais eficiente no controle de M. incognita no tomateiro. Os produtos biológicos e as diferentes doses de NemOut™ não afetaram o desenvolvimento das plantas de tomate.

Cell wall composition plays a key role on sensitivity of filamentous fungi to chitosan.

Chitosan antifungal activity has been reported for both filamentous fungi and yeast. Previous studies have shown fungal plasma membrane as main chitosan target. However, the role of the fungal cell wall (CW) in their response to chitosan is unknown. We show that cell wall regeneration in Neurospora crassa (chitosan sensitive) protoplasts protects them from chitosan damage. Caspofungin, a ß-1,3-glucan synthase inhibitor, showed a synergistic antifungal effect with chitosan for N. crassa but not for Pochonia chlamydosporia, a biocontrol fungus resistant to chitosan. Chitosan significantly repressed N. crassa genes involved in ß-1,3-glucan synthesis (fks) and elongation (gel-1) but the chitin synthase gene (chs-1) did not present changes in its expression. N. crassa cell wall deletion strains related to ß-1,3-glucan elongation (Δgel-1 and Δgel-2) were more sensitive to chitosan than wild type (wt). On the contrary, chitin synthase deletion strain (Δchs-1) showed the same sensitivity to chitosan than wt. The mycelium of P. chlamydosporia showed a higher (ca. twofold) ß-1,3-glucan/chitin ratio than that of N. crassa. Taken together, our results indicate that cell wall composition plays an important role on -sensitivity of filamentous fungi to chitosan.