Correction: Lozano-Soria et al. Volatile Organic Compounds from Entomopathogenic and Nematophagous Fungi, Repel Banana Black Weevil (Cosmopolites sordidus). Insects 2020, 11, 509.

In the original publication [...].

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.

Brindley's Glands Volatilome of the Predator Zelus renardii Interacting with Xylella Vectors.

Alien species must adapt to new biogeographical regions to acclimatise and survive. We consider a species to have become invasive if it establishes negative interactions after acclimatisation. Xylella fastidiosa Wells, Raju et al., 1986 (XF) represents Italy's and Europe's most recent biological invasion. In Apulia (southern Italy), the XF-encountered Philaenus spumarius L. 1758 (Spittlebugs, Hemiptera: Auchenorrhyncha) can acquire and transmit the bacterium to Olea europaea L., 1753. The management of XF invasion involves various transmission control means, including inundative biological control using Zelus renardii (ZR) Kolenati, 1856 (Hemiptera: Reduviidae). ZR is an alien stenophagous predator of Xylella vectors, recently entered from the Nearctic and acclimated in Europe. Zelus spp. can secrete semiochemicals during interactions with conspecifics and prey, including volatile organic compounds (VOCs) that elicit conspecific defence behavioural responses. Our study describes ZR Brindley's glands, present in males and females of ZR, which can produce semiochemicals, eliciting conspecific behavioural responses. We scrutinised ZR secretion alone or interacting with P. spumarius. The ZR volatilome includes 2-methyl-propanoic acid, 2-methyl-butanoic acid, and 3-methyl-1-butanol, which are consistent for Z. renardii alone. Olfactometric tests show that these three VOCs, individually tested, generate an avoidance (alarm) response in Z. renardii. 3-Methyl-1-butanol elicited the highest significant repellence, followed by 2-methyl-butanoic and 2-methyl-propanoic acids. The concentrations of the VOCs of ZR decrease during the interaction with P. spumarius. We discuss the potential effects of VOC secretions on the interaction of Z. renardii with P. spumarius.

Chitosan Modulates Volatile Organic Compound Emission from the Biocontrol Fungus Pochonia chlamydosporia.

Fungal volatile organic compounds (VOCs) are responsible for fungal odor and play a key role in biological processes and ecological interactions. VOCs represent a promising area of research to find natural metabolites for human exploitation. Pochonia chlamydosporia is a chitosan-resistant nematophagous fungus used in agriculture to control plant pathogens and widely studied in combination with chitosan. The effect of chitosan on the production of VOCs from P. chlamydosporia was analyzed using gas chromatography-mass spectrometry (GC-MS). Several growth stages in rice culture medium and different times of exposure to chitosan in modified Czapek-Dox broth cultures were analyzed. GC-MS analysis resulted in the tentative identification of 25 VOCs in the rice experiment and 19 VOCs in the Czapek-Dox broth cultures. The presence of chitosan in at least one of the experimental conditions resulted in the de novo production of 3-methylbutanoic acid and methyl 2,4-dimethylhexanoate, and oct-1-en-3-ol and tetradec-1-ene in the rice and Czapek-Dox experiments, respectively. Other VOCs changed their abundance because of the effect of chitosan and fungal age. Our findings suggest that chitosan can be used as a modulator of the production of VOCs in P. chlamydosporia and that there is also an effect of fungal age and exposure time.

"Ectomosphere": Insects and Microorganism Interactions.

This study focuses on interacting with insects and their ectosymbiont (lato sensu) microorganisms for environmentally safe plant production and protection. Some cases help compare ectosymbiont microorganisms that are insect-borne, -driven, or -spread relevant to endosymbionts' behaviour. Ectosymbiotic bacteria can interact with insects by allowing them to improve the value of their pabula. In addition, some bacteria are essential for creating ecological niches that can host the development of pests. Insect-borne plant pathogens include bacteria, viruses, and fungi. These pathogens interact with their vectors to enhance reciprocal fitness. Knowing vector-phoront interaction could considerably increase chances for outbreak management, notably when sustained by quarantine vector ectosymbiont pathogens, such as the actual Xylella fastidiosa Mediterranean invasion episode. Insect pathogenic viruses have a close evolutionary relationship with their hosts, also being highly specific and obligate parasites. Sixteen virus families have been reported to infect insects and may be involved in the biological control of specific pests, including some economic weevils. Insects and fungi are among the most widespread organisms in nature and interact with each other, establishing symbiotic relationships ranging from mutualism to antagonism. The associations can influence the extent to which interacting organisms can exert their effects on plants and the proper management practices. Sustainable pest management also relies on entomopathogenic fungi; research on these species starts from their isolation from insect carcasses, followed by identification using conventional light or electron microscopy techniques. Thanks to the development of omics sciences, it is possible to identify entomopathogenic fungi with evolutionary histories that are less-shared with the target insect and can be proposed as pest antagonists. Many interesting omics can help detect the presence of entomopathogens in different natural matrices, such as soil or plants. The same techniques will help localize ectosymbionts, localization of recesses, or specialized morphological adaptation, greatly supporting the robust interpretation of the symbiont role. The manipulation and modulation of ectosymbionts could be a more promising way to counteract pests and borne pathogens, mitigating the impact of formulates and reducing food insecurity due to the lesser impact of direct damage and diseases. The promise has a preventive intent for more manageable and broader implications for pests, comparing what we can obtain using simpler, less-specific techniques and a less comprehensive approach to Integrated Pest Management (IPM).

Beauveria bassiana (Hypocreales: Clavicipitaceae) Volatile Organic Compounds (VOCs) Repel Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae).

The entomopathogenic fungus Beauveria bassiana (Bb) is used to control the red palm weevil (RPW) Rhyncophorus ferrugineus (Oliver). Beuveria bassiana can infect and kill all developmental stages of RPW. We found that a solid formulate of B. bassiana isolate 203 (Bb203; CBS 121097), obtained from naturally infected RPW adults, repels RPW females. Fungi, and entomopathogens in particular, can produce volatile organic compounds (VOCs). VOCs from Bb203 were analyzed using gas chromatography-mass spectrometry (GC-MS). GC-MS identified more than 15 VOCs in B. bassiana not present in uninoculated (control) formulate. Both ethenyl benzene and benzothiazole B. bassiana VOCs can repel RPW females. Our findings suggest that B. bassiana and its VOCs can be used for sustainable management of RPW. They could act complementarily to avoid RPW infestation in palms.

Chitosan induces differential transcript usage of chitosanase 3 encoding gene (csn3) in the biocontrol fungus Pochonia chlamydosporia 123.

BACKGROUND: Pochonia chlamydosporia is an endophytic fungus used for nematode biocontrol that employs its cellular and molecular machinery to degrade the nematode egg-shell. Chitosanases, among other enzymes, are involved in this process. In this study, we improve the genome sequence assembly of P. chlamydosporia 123, by utilizing long Pacific Biosciences (PacBio) sequence reads. Combining this improved genome assembly with previous RNA-seq data revealed alternative isoforms of a chitosanase in the presence of chitosan. This study could open new insights into understanding fungal resistance to chitosan and root-knot nematode (RKN) egg infection processes. RESULTS: The P. chlamydosporia 123 genome sequence assembly has been updated using long-read PacBio sequencing and now includes 12,810 predicted protein-coding genes. Compared with the previous assembly based on short reads, there are 701 newly annotated genes, and 69 previous genes are now split. Eight of the new genes were differentially expressed in fungus interactions with Meloidogyne javanica eggs or chitosan. A survey of the RNA-seq data revealed alternative splicing in the csn3 gene that encodes a chitosanase, with four putative splicing variants: csn3_v1, csn3_v2, csn3_v3 and csn3_v4. When P. chlamydosporia is treated with 0.1 mg·mL- 1 chitosan for 4 days, csn3 is expressed 10-fold compared with untreated controls. Furthermore, the relative abundances of each of the four transcripts are different in chitosan treatment compared with controls. In controls, the abundances of each transcript are nil, 32, 55, and 12% for isoforms csn3_v1, csn3_v2, csn3_v3 and csn3_v4 respectively. Conversely, in chitosan-treated P. chlamydosporia, the abundances are respectively 80, 15%, 2-3%, 2-3%. Since isoform csn3_v1 is expressed with chitosan only, the putatively encoded enzyme is probably induced and likely important for chitosan degradation. CONCLUSIONS: Alternative splicing events have been discovered and described in the chitosanase 3 encoding gene from P. chlamydosporia 123. Gene csn3 takes part in RKN parasitism process and chitosan enhances its expression. The isoform csn3_v1 would be related to the degradation of this polymer in bulk form, while other isoforms may be related to the degradation of chitosan in the nematode egg-shell.

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.

Strain Degeneration in Pleurotus ostreatus: A Genotype Dependent Oxidative Stress Process Which Triggers Oxidative Stress, Cellular Detoxifying and Cell Wall Reshaping Genes.

Strain degeneration has been defined as a decrease or loss in the yield of important commercial traits resulting from subsequent culture, which ultimately leads to Reactive Oxygen Species (ROS) production. Pleurotus ostreatus is a lignin-producing nematophagous edible mushroom. Mycelia for mushroom production are usually maintained in subsequent culture in solid media and frequently show symptoms of strain degeneration. The dikaryotic strain P. ostreatus (DkN001) has been used in our lab as a model organism for different purposes. Hence, different tools have been developed to uncover genetic and molecular aspects of this fungus. In this work, strain degeneration was studied in a full-sib monokaryotic progeny of the DkN001 strain with fast (F) and slow (S) growth rates by using different experimental approaches (light microscopy, malondialdehyde levels, whole-genome transcriptome analysis, and chitosan effect on monokaryotic mycelia). The results obtained showed that: (i) strain degeneration in P. ostreatus is linked to oxidative stress, (ii) the oxidative stress response in monokaryons is genotype dependent, (iii) stress and detoxifying genes are highly expressed in S monokaryons with symptoms of strain degeneration, (iv) chitosan addition to F and S monokaryons uncovered the constitutive expression of both oxidative stress and cellular detoxifying genes in S monokaryon strains which suggest their adaptation to oxidative stress, and (v) the overexpression of the cell wall genes, Uap1 and Cda1, in S monokaryons with strain degeneration phenotype indicates cell wall reshaping and the activation of High Osmolarity Glycerol (HOG) and Cell Wall Integrity (CWI) pathways. These results could constitute a hallmark for mushroom producers to distinguish strain degeneration in commercial mushrooms.

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.

Chitosan inhibits septin-mediated plant infection by the rice blast fungus Magnaporthe oryzae in a protein kinase C and Nox1 NADPH oxidase-dependent manner.

Chitosan is a partially deacetylated linear polysaccharide composed of ß-1,4-linked units of d-glucosamine and N-acetyl glucosamine. As well as a structural component of fungal cell walls, chitosan is a potent antifungal agent. However, the mode of action of chitosan is poorly understood. Here, we report that chitosan is effective for control of rice blast disease. Chitosan application impairs growth of the blast fungus Magnaporthe oryzae and has a pronounced effect on appressorium-mediated plant infection. Chitosan inhibits septin-mediated F-actin remodelling at the appressorium pore, thereby preventing repolarization of the infection cell. Chitosan causes plasma membrane permeabilization of M. oryzae and affects NADPH oxidase-dependent synthesis of reactive oxygen species, essential for septin ring formation and fungal pathogenicity. We further show that toxicity of chitosan to M. oryzae requires the protein kinase C-dependent cell wall integrity pathway, the Mps1 mitogen-activated protein kinase and the Nox1 NADPH oxidase. A conditionally lethal, analogue (PP1)-sensitive mutant of Pkc1 is partially remediated for growth in the presence of chitosan, while ∆nox1 mutants increase their glucan : chitin cell wall ratio, rendering them resistant to chitosan. Taken together, our data show that chitosan is a potent fungicide which requires the cell integrity pathway, disrupts plasma membrane function and inhibits septin-mediated plant infection.

Chitosan modulates Pochonia chlamydosporia gene expression during nematode egg parasitism.

Climate change makes plant-parasitic nematodes (PPN) an increasing threat to commercial crops. PPN can be managed sustainably by the biocontrol fungus Pochonia chlamydosporia (Pc). Chitosan generated from chitin deacetylation enhances PPN parasitism by Pc. In this work, we investigate the molecular mechanisms of Pc for chitosan resistance and root-knot nematode (RKN) parasitism, using transcriptomics. Chitosan and RKN modify the expression of Pc genes, mainly those involved in oxidation-reduction processes. Both agents significantly modify the expression of genes associated to 113 GO terms and 180 Pc genes. Genes encoding putative glycoproteins (Pc adhesives) to nematode eggshell, as well as genes involved in redox, carbohydrate and lipid metabolism trigger the response to chitosan. We identify genes expressed in both the parasitic and endophytic phases of the Pc lifecycle; these include proteases, chitosanases and transcription factors. Using the Pathogen-Host Interaction database (PHI-base), our previous RNA-seq data and RT-PCR of Pc colonizing banana we have investigated genes expressed both in the parasitic and endophytic phases of Pc lifecycle.

Chitosan Induces Plant Hormones and Defenses in Tomato Root Exudates.

In this work, we use electrophysiological and metabolomic tools to determine the role of chitosan as plant defense elicitor in soil for preventing or manage root pests and diseases sustainably. Root exudates include a wide variety of molecules that plants and root microbiota use to communicate in the rhizosphere. Tomato plants were treated with chitosan. Root exudates from tomato plants were analyzed at 3, 10, 20, and 30 days after planting (dap). We found, using high performance liquid chromatography (HPLC) and excitation emission matrix (EEM) fluorescence, that chitosan induces plant hormones, lipid signaling and defense compounds in tomato root exudates, including phenolics. High doses of chitosan induce membrane depolarization and affect membrane integrity. 1H-NMR showed the dynamic of exudation, detecting the largest number of signals in 20 dap root exudates. Root exudates from plants irrigated with chitosan inhibit ca. twofold growth kinetics of the tomato root parasitic fungus Fusarium oxysporum f. sp. radicis-lycopersici. and reduced ca. 1.5-fold egg hatching of the root-knot nematode Meloidogyne javanica.

Volatile Organic Compounds from Entomopathogenic and Nematophagous Fungi, Repel Banana Black Weevil (Cosmopolites sordidus).

Fungal Volatile Organic Compounds (VOCs) repel banana black weevil (BW), Cosmopolites sordidus (Germar, 1824), the key-pest of banana [Musa sp. (Linnaeus, 1753)]. The entomopathogens Beauveria bassiana (Bb1TS11) and Metarhizium robertsii (Mr4TS04) were isolated from banana plantation soils using an insect bait. Bb1TS11 and Mr4TS04 were pathogenic to BW adults. Bb1TS11, Bb203 (from infected palm weevils), Mr4TS04 and the nematophagous fungus Pochonia clamydosporia (Pc123), were tested for VOCs production. VOCs were identified by Gas Chromatography/Mass Spectrometry-Solid-Phase Micro Extraction (GC/MS-SPME). GC/MS-SPME identified a total of 97 VOCs in all strains tested. Seven VOCs (styrene, benzothiazole, camphor, borneol, 1,3-dimethoxy-benzene, 1-octen-3-ol and 3-cyclohepten-1-one) were selected for their abundance or previous record as insect repellents. BW-starved adults in the dark showed the highest mobility to banana corm in olfactometry bioassays. 3-cyclohepten-1-one (C7), produced by all fungal strains, is the best BW repellent (p < 0.05), followed by 1,3-dimethoxy-benzene (C5). The rest of the VOCs have a milder repellency to BW. Styrene (C1) and benzothiazole (C2) (known to repel palm weevil) block the attraction of banana corm and BW pheromone to BW adults in bioassays. Therefore, VOCs from biocontrol fungi can be used in future studies for the biomanagement of BW in the field.

Molecular Mechanisms of Chitosan Interactions with Fungi and Plants.

Chitosan is a versatile compound with multiple biotechnological applications. This polymer inhibits clinically important human fungal pathogens under the same carbon and nitrogen status as in blood. Chitosan permeabilises their high-fluidity plasma membrane and increases production of intracellular oxygen species (ROS). Conversely, chitosan is compatible with mammalian cell lines as well as with biocontrol fungi (BCF). BCF resistant to chitosan have low-fluidity membranes and high glucan/chitin ratios in their cell walls. Recent studies illustrate molecular and physiological basis of chitosan-root interactions. Chitosan induces auxin accumulation in Arabidopsis roots. This polymer causes overexpression of tryptophan-dependent auxin biosynthesis pathway. It also blocks auxin translocation in roots. Chitosan is a plant defense modulator. Endophytes and fungal pathogens evade plant immunity converting chitin into chitosan. LysM effectors shield chitin and protect fungal cell walls from plant chitinases. These enzymes together with fungal chitin deacetylases, chitosanases and effectors play determinant roles during fungal colonization of plants. This review describes chitosan mode of action (cell and gene targets) in fungi and plants. This knowledge will help to develop chitosan for agrobiotechnological and medical applications.

Genome and secretome analysis of Pochonia chlamydosporia provide new insight into egg-parasitic mechanisms.

Pochonia chlamydosporia infects eggs and females of economically important plant-parasitic nematodes. The fungal isolates parasitizing different nematodes are genetically distinct. To understand their intraspecific genetic differentiation, parasitic mechanisms, and adaptive evolution, we assembled seven putative chromosomes of P. chlamydosporia strain 170 isolated from root-knot nematode eggs (~44 Mb, including 7.19% of transposable elements) and compared them with the genome of the strain 123 (~41 Mb) isolated from cereal cyst nematode. We focus on secretomes of the fungus, which play important roles in pathogenicity and fungus-host/environment interactions, and identified 1,750 secreted proteins, with a high proportion of carboxypeptidases, subtilisins, and chitinases. We analyzed the phylogenies of these genes and predicted new pathogenic molecules. By comparative transcriptome analysis, we found that secreted proteins involved in responses to nutrient stress are mainly comprised of proteases and glycoside hydrolases. Moreover, 32 secreted proteins undergoing positive selection and 71 duplicated gene pairs encoding secreted proteins are identified. Two duplicated pairs encoding secreted glycosyl hydrolases (GH30), which may be related to fungal endophytic process and lost in many insect-pathogenic fungi but exist in nematophagous fungi, are putatively acquired from bacteria by horizontal gene transfer. The results help understanding genetic origins and evolution of parasitism-related genes.

Induction of auxin biosynthesis and WOX5 repression mediate changes in root development in Arabidopsis exposed to chitosan.

Chitosan is a natural polymer with applications in agriculture, which causes plasma membrane permeabilisation and induction of intracellular reactive oxygen species (ROS) in plants. Chitosan has been mostly applied in the phylloplane to control plant diseases and to enhance plant defences, but has also been considered for controlling root pests. However, the effect of chitosan on roots is virtually unknown. In this work, we show that chitosan interfered with auxin homeostasis in Arabidopsis roots, promoting a 2-3 fold accumulation of indole acetic acid (IAA). We observed chitosan dose-dependent alterations of auxin synthesis, transport and signalling in Arabidopsis roots. As a consequence, high doses of chitosan reduce WOX5 expression in the root apical meristem and arrest root growth. Chitosan also propitiates accumulation of salicylic (SA) and jasmonic (JA) acids in Arabidopsis roots by induction of genes involved in their biosynthesis and signalling. In addition, high-dose chitosan irrigation of tomato and barley plants also arrests root development. Tomato root apices treated with chitosan showed isodiametric cells respect to rectangular cells in the controls. We found that chitosan causes strong alterations in root cell morphology. Our results highlight the importance of considering chitosan dose during agronomical applications to the rhizosphere.

Chitosan Increases Tomato Root Colonization by Pochonia chlamydosporia and Their Combination Reduces Root-Knot Nematode Damage.

The use of biological control agents could be a non-chemical alternative for management of Meloidogyne spp. [root-knot nematodes (RKN)], the most damaging plant-parasitic nematodes for horticultural crops worldwide. Pochonia chlamydosporia is a fungal parasite of RKN eggs that can colonize endophytically roots of several cultivated plant species, but in field applications the fungus shows a low persistence and efficiency in RKN management. The combined use of P. chlamydosporia with an enhancer could help its ability to develop in soil and colonize roots, thereby increasing its efficiency against nematodes. Previous work has shown that chitosan enhances P. chlamydosporia sporulation and production of extracellular enzymes, as well as nematode egg parasitism in laboratory bioassays. This work shows that chitosan at low concentrations (up to 0.1 mg ml-1) do not affect the viability and germination of P. chlamydosporia chlamydospores and improves mycelial growth respect to treatments without chitosan. Tomato plants irrigated with chitosan (same dose limit) increased root weight and length after 30 days. Chitosan irrigation increased dry shoot and fresh root weight of tomato plants inoculated with Meloidogyne javanica, root length when they were inoculated with P. chlamydosporia, and dry shoot weight of plants inoculated with both P. chlamydosporia and M. javanica. Chitosan irrigation significantly enhanced root colonization by P. chlamydosporia, but neither nematode infection per plant nor fungal egg parasitism was affected. Tomato plants cultivated in a mid-suppressive (29.3 ± 4.7% RKN egg infection) non-sterilized clay loam soil and irrigated with chitosan had enhanced shoot growth, reduced RKN multiplication, and disease severity. Chitosan irrigation in a highly suppressive (73.7 ± 2.6% RKN egg infection) sterilized-sandy loam soil reduced RKN multiplication in tomato. However, chitosan did not affect disease severity or plant growth irrespective of soil sterilization. Chitosan, at an adequate dose, can be a potential tool for sustainable management of RKN.

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.

Tolerance to chitosan by Trichoderma species is associated with low membrane fluidity.

The effect of chitosan on growth of Trichoderma spp., a cosmopolitan genus widely exploited for their biocontrol properties was evaluated. Based on genotypic (ITS of 18S rDNA) characters, four isolates of Trichoderma were identified as T. pseudokoningii FLM16, T. citrinoviride FLM17, T. harzianum EZG47, and T. koningiopsis VSL185. Chitosan reduces radial growth of Trichoderma isolates in concentration-wise manner. T. koningiopsis VSL185 was the most chitosan tolerant isolate in all culture media amended with chitosan (0.5-2.0 mg ml(-1) ). Minimal Inhibitory Concentration (MIC) and Minimal Fungicidal Concentration (MFC) were determined showing that T. koningiopsis VSL185 displays higher chitosan tolerance with MIC value >2000 µg ml(-1) while for other Trichoderma isolates MIC values were around 10 µg ml(-1) . Finally, free fatty acid composition reveals that T. koningiopsis VSL185, chitosan tolerant isolate, displays lower linolenic acid (C18:3) content than chitosan sensitive Trichoderma isolates. Our findings suggest that low membrane fluidity is associated with chitosan tolerance in Trichoderma spp.