Federico Lopez-Moya

Carbon and nitrogen limitation increase chitosan antifungal activity in Neurospora crassa and fungal human pathogens

Chitosan permeabilizes plasma membrane and kills sensitive filamentous fungi and yeast. Membrane fluidity and cell energy determine chitosan sensitivity in fungi. A five-fold reduction of both glucose (main carbon (C) source) and nitrogen (N) increased 2-fold Neurospora crassa sensitivity to chitosan. We linked this increase with production of intracellular reactive oxygen species (ROS) and plasma membrane permeabilization. Releasing N. crassa from nutrient limitation reduced chitosan antifungal activity in spite of high ROS intracellular levels. With lactate instead of glucose, C and N limitation increased N. crassa sensitivity to chitosan further (4-fold) than what glucose did. Nutrient limitation also increased sensitivity of filamentous fungi and yeast human pathogens to chitosan. For Fusarium proliferatum, lowering 100-fold C and N content in the growth medium, increased 16-fold chitosan sensitivity. Similar results were found for Candida spp. (including fluconazole resistant strains) and Cryptococcus spp. Severe C and N limitation increased chitosan antifungal activity for all pathogens tested. Chitosan at 100 μg ml(-1) was lethal for most fungal human pathogens tested but non-toxic to HEK293 and COS7 mammalian cell lines. Besides, chitosan increased 90% survival of Galleria mellonella larvae infected with C. albicans. These results are of paramount for developing chitosan as antifungal.

Chitosan enhances parasitism of Meloidogyne javanica eggs by the nematophagous fungus Pochonia chlamydosporia.

Pochonia chlamydosporia (Pc), a nematophagous fungus and root endophyte, uses appressoria and extracellular enzymes, principally proteases, to infect the eggs of plant parasitic nematodes (PPN). Unlike other fungi, Pc is resistant to chitosan, a deacetylated form of chitin, used in agriculture as a biopesticide to control plant pathogens. In the present work, we show that chitosan increases Meloidogyne javanica egg parasitism by P. chlamydosporia. Using antibodies specific to the Pc enzymes VCP1 (a subtilisin), and SCP1 (a serine carboxypeptidase), we demonstrate chitosan elicitation of the fungal proteases during the parasitic process. Chitosan increases VCP1 immuno-labelling in the cell wall of Pc conidia, hyphal tips of germinating spores, and in appressoria on infected M. javanica eggs. These results support the role of proteases in egg parasitism by the fungus and their activation by chitosan. Phylogenetic analysis of the Pc genome reveals a large diversity of subtilisins (S8) and serine carboxypeptidases (S10). The VCP1 group in the S8 tree shows evidence of gene duplication indicating recent adaptations to nutrient sources. Our results demonstrate that chitosan enhances Pc infectivity of nematode eggs through increased proteolytic activities and appressoria formation and might be used to improve the efficacy of M. javanica biocontrol.

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 β‐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 β‐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 β‐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) β‐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.

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.

Omics for Investigating Chitosan as an Antifungal and Gene Modulator

Chitosan is a biopolymer with a wide range of applications. The use of chitosan in clinical medicine to control infections by fungal pathogens such as Candida spp. is one of its most promising applications in view of the reduced number of antifungals available. Chitosan increases intracellular oxidative stress, then permeabilizes the plasma membrane of sensitive filamentous fungus Neurospora crassa and yeast. Transcriptomics reveals plasma membrane homeostasis and oxidative metabolism genes as key players in the response of fungi to chitosan. A lipase and a monosaccharide transporter, both inner plasma membrane proteins, and a glutathione transferase are main chitosan targets in N. crassa. Biocontrol fungi such as Pochonia chlamydosporia have a low content of polyunsaturated free fatty acids in their plasma membranes and are resistant to chitosan. Genome sequencing of P. chlamydosporia reveals a wide gene machinery to degrade and assimilate chitosan. Chitosan increases P. chlamydosporia sporulation and enhances parasitism of plant parasitic nematodes by the fungus. Omics studies allow understanding the mode of action of chitosan and help its development as an antifungal and gene modulator.

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.

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.

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.

Pochonia chlamydosporia : Multitrophic Lifestyles Explained by a Versatile Genome

The nematophagous fungus Pochonia chlamydosporia (Goddard) Zare & Gams is a facultative parasite of nematode females and eggs. This fungus has a world-wide distribution and has the capacity to survive in the absence of the nematodes. Pochonia chlamydosporia is rhizosphere competent and can colonize the rhizosphere of crops of economic importance, such as tomato and barley. The infection of nematode eggs by P. chlamydosporia requires adhesion, differentiation of appressoria, and penetration of their egg shells. Since the 1980s, proteases have been described as the main group of enzymes that the fungus uses to penetrate nematode eggs. Recent studies found that these genes are also expressed when the fungus endophytically colonizes barley roots. The genome of the fungus was sequenced in 2014, this being the first publicly available genome of a fungus parasite of nematode eggs. Since then, many publications based on the fungus genome have been published. Study of the major gene families in the genome shows the tools that the fungus uses in its different lifestyles. Transcriptomic studies using DNA microarrays and RNA-seq have been done to study the molecular mechanisms that regulate endophytic colonization of Pochonia chlamydosporia. A general analysis of the genome allowed us to identify a large number of carbohydrate active enzymes (Cazy) which help the fungus in colonizing plants and infecting nematodes.