Ernesto A. Zavala-Gonzalez

CAZyme content of Pochonia chlamydosporia reflects that chitin and chitosan modification are involved in nematode parasitism

Pochonia chlamydosporia is a soil fungus with a multitrophic lifestyle combining endophytic and saprophytic behaviors, in addition to a nematophagous activity directed against eggs of root-knot and other plant parasitic nematodes. The carbohydrate-active enzymes encoded by the genome of P. chlamydosporia suggest that the endophytic and saprophytic lifestyles make use of a plant cell wall polysaccharide degradation machinery that can target cellulose, xylan and, to a lesser extent, pectin. This enzymatic machinery is completed by a chitin breakdown system that involves not only chitinases, but also chitin deacetylases and a large number of chitosanases. P. chlamydosporia can degrade and grow on chitin and is particularly efficient on chitosan. The relevance of chitosan breakdown during nematode egg infection is supported by the immunolocalization of chitosan in Meloidogyne javanica eggs infected by P. chlamydosporia and by the fact that the fungus expresses chitosanase and chitin deacetylase genes during egg infection. This suggests that these enzymes are important for the nematophagous activity of the fungus and they are targets for improving the capabilities of P. chlamydosporia as a biocontrol agent in agriculture.

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.

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.

Ethanol production from chitosan by the nematophagous fungus Pochonia chlamydosporia and the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana

Chitin is the second most abundant biopolymer after cellulose and virtually unexplored as raw material for bioethanol production. In this paper, we investigate chitosan, the deacetylated form of chitin which is the main component of shellfish waste, as substrate for bioethanol production by fungi. Fungal parasites of invertebrates such as the nematophagous Pochonia chlamydosporia (Pc) or the entomopathogens Beauveria bassiana (Bb) and Metarhizium anisopliae (Ma) are biocontrol agents of plant parasitic nematodes (eg. Meloidogyne spp.) or insect pests such as the red palm weevil (Rhynchophorus ferrugineus). These fungi degrade chitin-rich barriers for host penetration. We have therefore tested the chitin/chitosanolytic capabilities of Pc, Bb and Ma for generating reducing sugars using chitosan as only nutrient. Among the microorganisms used in this study, Pc is the best chitosan degrader, even under anaerobic conditions. These fungi have alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) encoding genes in their genomes. We have therefore analyzed their ethanol production under anaerobic conditions using chitosan as raw material. P. chlamydosporia is the largest ethanol producer from chitosan. Our studies are a starting point to develop chitin-chitosan based biofuels.

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.