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.This work was supported by Spanish Ministry of Economy and Competitiveness Grant AGL 2015 66833-R and a sabbatical grant (PR2015-00087) to Luis V. Lopez-Llorca

Modulation of the Host Defence System by Nematophagous Fungi and Chitosan

Nematophagous fungi (NFs), which are responsible for soil suppression of plant-parasitic nematodes, are multitrophic biocontrol agents. This raises the question of the transition between lifestyles (e.g., endophytism vs. egg parasitism). The NF Pochonia chlamydosporia colonises food crops and promotes their growth and yield. When colonising the plant, P. chlamydosporia induces the plant immunity (PI). However, it also evades the PI. To do this, both endophytic NF and pathogenic fungi (PF) secrete LysM effectors (LysM-effs). LysM effectors have been shown to have dverse functions in different organisms, including the protection of fungal chitin from plant chitinases. P. chlamydosporia is resistant to chitosan, which modulates gene expression in fungi and plants and has antimicrobial properties. P. chlamydosporia chitin deacetylases (CDA) and chitosanases (CSN) also help P. chlamydosporia evade plant immunity, resist exogenous chitosan, and are induced during fungal infection of nematode eggs. NF-chitosan formulations are new biomanagement tools against plant parasitic nematodes, fungal wilt pathogens and insect pests that currently threaten food security crops. Furthermore, omics techniques are useful tools to elucidate the role of CDAs, CSNs, LysM-effs, adhesion proteins and carbohydrate-active enzymes in pathogen–BCA–plant interactions, adhesión and infection to nematode eggs and their modulation by chitosan.This project has been funded by the PID-2020-119734RB-I00 project of the Spanish Ministry of Science and Innovation

Isolates of the Nematophagous Fungus Pochonia chlamydosporia Are Endophytic in Banana Roots and Promote Plant Growth

The biocontrol fungus Pochonia chlamydosporia colonizes banana roots endophytically. Root hairs and root surface were colonized by a stable GFP (green fluorescent protein) transformant of the fungus. Hyphal penetration in root cells was also observed. Spores of P. chlamydosporia 123, significantly increase root and leaf length and weight in banana plantlets (Musa acuminata cv. ‘Dwarf Cavendish’) in growth chamber experiments 30 days post-inoculation. In greenhouse 8-L pot experiments, P. chlamydosporia 123 spore inoculation significantly increases root, corm and leaf length, and leaf weight in banana plants (75 days post-inoculation). Spore inoculation of P. chlamydosporia strains from diverse origin (Pc21, Pc123, Pc399, and Pccat), significantly increase root, corm and leaf length and weight in banana plantlets. Pc21 from Italy was the best colonizer of banana roots. Consequently, this strain significantly increases banana root and leaf length most. Root colonization by P. chlamydosporia was also detected using cultural techniques and qPCR.This research was funded by EU H2020, Musa Project (727624)

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.This research was funded by PID2020-119734RB-I00 Project from the Spanish Ministry of Science and Innovation and by European Project H2020 MUSA no. 727624

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.This research was funded by European Project H2020 MUSA, Grant Number 727624

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.This work was supported by the Spanish Ministry of Science and Innovation AGL2015-66833-R project, the Universiti Kebangsaan Malaysia, and the Malaysia Ministry of Higher Education. Project was also partially supported by MUSA project (727624). The results of this paper have been filed for a Spanish Patent (P201631534)

On the Presence and Ubiquity of the Exotic Batophora (J. Agardh) in the Mar Menor Lagoon (SE Spain)

We first report the presence of a species of the genus Batophora in the Iberian Peninsula, in the Mar Menor lagoon (Murcia, SE Spain). We detected this macroalga in November 2021. However, according to some observations, it could have been present in the lagoon at least since 2016, being described as a “rare” form of Dasycladus vermicularis. A further survey made during May 2022 confirmed the presence of this species in much of the perimeter of the Mar Menor. Morphologic analyses are not conclusive regarding the species description, but most traits point to Batophora occidentalis or Batophora occidentalis var. largoensis. A consensus sequence from the rbcL barcode gene was compared by the Blastn tool showing its closeness to other Batophora specimens, yet the specific level was unresolved. The recent confirmation of a Batophora species in a littoral lagoon from Formentera (Balearic Islands) with similar morphologic characteristics could indicate that both putative introductions are connected. Further morphologic and genetic analyses are required in order to know the origin and expansion of this genus along the Mediterranean coasts

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.This research was funded by H2020 European Project Microbial Uptakes for Sustainable management of major bananA pests and diseases with project number 727624

Detection of Haplosporidium pinnae from Pinna nobilis Faeces

Pinna nobilis (Linnaeus, 1758) is the largest bivalve endemic to the Mediterranean. It is distributed in a wide range of coastal environments, including estuaries. Pinna nobilis has recently become a critically endangered species (with almost 100% mortality) along the entire Spanish Mediterranean coast. This may be due to coinfections caused by Haplosporidium pinnae and bacterial pathogens such as Mycobacterium spp. We extensively sampled P. nobilis from Mar Menor lagoon (SE Spain), a site where individuals still survive. Using conventional PCR, we found Haplosporidium spp. in 7.1% of mantle and faecal DNA samples in different individuals of P. nobilis. We identified and quantified Haplosporidium pinnae in P. nobilis using Sanger sequencing and qPCR. Faecal H. pinnae detection is non-invasive, unlike biopsies. Therefore, this non-lethal and non-invasive sampling method could contribute to the welfare of living populations, particularly in eutrophic environments, where they are prone to septicaemia. The use of faecal DNA analysis could be a major advance in epidemiology and recovery assessment studies of P. nobilis.info:eu-repo/semantics/publishedVersio

Multidisciplinary Analysis of Cystoseira sensu lato (SE Spain) Suggest a Complex Colonization of the Mediterranean

Cystoseira sensu lato (sl) are three genera widely recognized as bioindicators for their restricted habitat in a sub-coastal zone with low tolerance to pollution. Their ecological, morphological and taxonomic features are still little known due to their singular characteristics. We studied seven species of Cystoseira sl spp. in Cabo de las Huertas (Alicante, SE Spain) and analyzed their distribution using Permutational Analysis of Variance (PERMANOVA) and Principal Component Ordination plots (PCO). A morphological cladogram has been constructed using fifteen phenotypic taxonomic relevant characters. We have also developed an optimized Cystoseira sl DNA extraction protocol. We have tested it to obtain amplicons from mt23S, tRNA-Lys and psbA genes. With these sequence data, we have built a phylogenetic supertree avoiding threatened Cystoseira sl species. Cartography and distribution analysis show that the response to hydrodynamism predicts perennial or seasonal behaviors. Morphological cladogram detects inter-specifical variability between our species and reference studies. Our DNA phylogenetic tree supports actual classification, including for the first-time Treptacantha sauvageauana and Treptacantha algeriensis species. These data support a complex distribution and speciation of Cystoseira sl spp. in the Mediterranean, perhaps involving Atlantic clades. The high ecological value of our area of study merits a future protection status as a Special Conservation Area.This research received partial funding from the Botany section (36502G0001) of the Department of Marine Science and Applied Biology, the University of Alicante