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.

SummaryPochonia 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 floweringtime 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.

Summary 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.

The fungal parasite of nematode eggs Pochonia chlamydosporia is also a root endophyte known to promote growth of some plants. In this study, we analysed the effect of nine P. chlamydosporia isolates from worldwide origin on tomato growth. Experiments were performed at different scales (Petri dish, growth chamber and greenhouse conditions) and developmental stages (seedlings, plantlets and plants). Seven P. chlamydosporia isolates significantly (P < 0.05) increased the number of secondary roots and six of those increased total weight of tomato seedlings. Six P. chlamydosporia isolates also increased root weight of tomato plantlets. Root colonisation varied between different isolates of this fungus. Again P. chlamydosporia significantly increased root growth of tomato plants under greenhouse conditions and reduced flowering and fruiting times (up to 5 and 12 days, respectively) versus uninoculated tomato plants. P. chlamydosporia increased mature fruit weight in tomato plants. The basis of the mechanisms for growth, flowering and yield promotion in tomato by the fungus are unknown. However, we found that P. chlamydosporia can produce Indole-3-acetic acid and solubilise mineral phosphate. These results suggest that plant hormones or nutrient ability could play an important role. Our results put forward the agronomic importance of P. chlamydosporia as biocontrol agent of plant parasitic nematodes with tomato growth promoting capabilities.

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.