Tesis sobre el tema "Mycoherbicide"

A foliar pathogen identified as a member of the genus Alternaria was isolated from blighted Sphenoclea zeylanica (gooseweed) collected in 1991 from a rice field near Los Banos, Laguna, Philippines. Inoculum density, dew period, and plant height are factors influencing biocontrol of S. zeylanica with this indigenous pathogen. Significantly higher percent reductions in plant height and dry weight were obtained and all plants were killed at higher inoculum concentrations with 8 h of dew. The number, germination, and virulence of conidia were significantly affected by production techniques, temperature, light condition, and incubation period. Exposure to continuous near-ultraviolet (NUV) light at 28$ sp circ$C stimulated sporulation on agar media and on solid substrates. Overall, the best production technique was the use of sorghum seeds using an equal quantity of sorghum seeds and water (w/v) incubated for four weeks. Another conidia production method using the sporulation medium (S-medium) technique was evaluated with the addition of 20 g L$ sp{-1}$ of calcium carbonate (CaCO$ sb3)$ and 2 ml of sterile distilled water. Primary 1/2 PDA at 18$ sp circ$C in the dark produced the most virulent conidia. This technique produced conidia relatively rapid, but was labour intensive. Host range studies using 49 plant species in 40 genera representing 20 families, selected by using a modified centrifugal phylogenetic and variety strategy indicated that only S. zeylanica was susceptible in the absence and presence of supplemental dew. On the basis of morphological and cultural characteristics, pathogenicity on the host, host specificity, and the absence of a previous record of this fungal pathogen on S. zeylanica, the binomial A. alternata f. sp. sphenocleae is proposed.

L’objectif de cette thèse était le développement d’un mycoherbicide efficace contenant les propagules stables de Plectosporium alismatis, un mycoherbicide potentiel de riz. Le champignon produit des conidies (109 L-1) et des chlamydospores (108 L-1) dans les erlens. Le nitrate est essentiel pour la production de chlamydospores qui ont une survie élevée (6. 6% germination après 4 mois) comparée aux conidies. Des agrégats hétérogènes (contenant les clilamydospores) ont été aussi observés en milieu liquide contenant du nitrate. Ces agrégats pouffaient augmenter la résistance de P. Aljymatis contre les stresses environnementaux. La production d’agrégats homogènes sphériques (les pellets) est nécessaire pour améliorer la reproductibilité. Un milieu « Aggregate Production Medium (APM) » a été développé dans lequel des pellets (~2. 8 x 105 L-1), des conidies (~6. 5 x 109 L-1) et des chlamydospores (~9. 6 x 108 L-1) ont été produits. Après la dessiccation et l’exposition aux UV, le nombre maximal de propagules libres (~104 conidies et ~2 x 102 chlamydospores) n’a pas infecté la feuille alors qu’un seul pellet était suffisant pour causer la nécrose. Nous avons donc conclu que ces pellets étaient plus pathogènes car probablement plus résistants aux UV et à la dessiccation que les conidies. Les études de survie ont montré que la formulation diatomite contenant des conidies libres, des chlamydospores libres et des pellets, stockée à 4°C, survivait après 9 mois de conservation (12% de germination) et restait pathogène. En conclusion, P. Alismatis peut produire des pellets en milieu APM, et la formulation diatomite contenant ces pellets pourrait répondre aux qualités exigées d’un mycoherbicide efficace
The objective of this thesis was to develop an efficient mycoherbicide based on the stable propagules produced by Plectosporium alismatis, a potential mycoherbicide in rice crops. The fungus produced conidia (109 L-1) and chlamydospores (108 L-1) in shake-flasks. Nitrate was required for production of chlamydospores which had a ligher shelf-life (6. 6% germination after 4 months) compared to conidia. The presence of aggregates (containing chlamydospores) ws also observed in nitrate-based medium. These aggregates may increase P. Alismatis resistance against environmental constraints. To overcome the lack of culture reproducibility due to aggregates’ heterogeneous nature, an alternative, the production of homogeneous spherical aggregates (i. E. , pellets) was developed. In a new medium “Aggregate Production Medium (APM)” in which up to 2. 8 x l05 pellets L-1, 6. 5 x l09 conidia L-1 and 9. 6 x 108 chlamydospores L-1 were produced. When maximal number of free propagules (~104 conidia and ~2 x 102 chlamydospores) was not able to infect leaf when dried and exposed to U, only a single pellet was required to induce disease in the same conditions. We conclude that pellets were far more pathogenic and tolerant to desiccation and UV compared to conidia. In shelf-life experiments, diatomite formulation containing free conidia, free chlamydospores and pellets, stored at 4°C survived for a long period (12% germination after 9 months) and remained pathogenic. In conclusion, P. Alismatis can produce pellets in APM, and diatomite formulations based on these pellet-containing cultures could satisfy the requirements for an efficient mycoherbicide

Amaranthus retroflexus L. (redroot pigweed) is a major weed of many crops in North America including corn, soybean, and potato. It can be readily controlled by chemical and cultural methods. However, some populations of A. retroflexus have developed resistance against the application of triazine herbicides. Biololical control could be an alternative method to control this weed species. In 1990, a Macrophoma sp. causing foliar lesions was isolated from redroot pigweed and the potential of this plant pathogenic fungus as a mycoherbicide was evaluated. Large numbers of infective propagules were produced in solid substrate fermentation with chickpeas. When inoculated with 10$ sp8$ or 10$ sp9$ conidia m$ sp{-2}$, plants at the cotyledon to 2-leaf stage showed the most severe damage. Disease developed over a wide range of dew period durations (6 hr to 24 hr) and temperature regimes (14 C to 26 C), and the most rapid and destructive disease development occurred following a 24-hr dew period at 18 C. In controlled environment studies, this Macrophoma sp. was pathogenic to the genus Amaranthus and the closely related genus Celosia.

Thesis (MSc)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Acacia cyclops, an invasive weed in South Africa, was initially imported to stabilize the sand dunes in the southern Cape. The spread of A. cyclops is a major threat to the fragile biodiversity of the strandveld and limestone fynbos vegetation. Acacia cyclops dieback has been observed for some time, although the causative agent, Pseudolagarobasidium acaciicola, has only recently been described. This fungus is nominated for development as a mycoherbicide to control A. cyclops. Although current biological and mechanical control efforts are proving to be partially effective, A. cyclops is still causing major damage to natural ecosystems. The introduction of a mycoherbicide would increase the cost effectiveness of controlling this weed in the long term. The majority of the literature that was reviewed supports the use of mycoherbicides as biocontrol agents, especially when taking into account the decrease in acceptance and availability of chemical control agents. Considering that the Pseudolagarobasidium genus consists of saprobes, opportunistic facultative pathogens and endophytes, P. acaciicola is predicted to have similar biological characteristics. The species is also highly likely to be indigenous, although with a wider distribution range than previously envisaged. Strict precautions should still however be taken to ensure that non-target species will not be threatened. This study consists of a unique risk assessment comprising different sections. A field survey was performed to record disease incidence among indigenous woody plant species around 100 diseased A. cyclops trees. Subsequently, DNA extractions were made from the roots of the diseased indigenous plants and A. cyclops trees to verify the presence of P. acaciicola. Of the 2432 indigenous woody plants observed, 22 (0.9%) were dead or dying, while P. acaciicola was detected in 10 of these (0.4%), representing six species. Pseudolagarobasidium acaciicola was detected in 47% of the A. cyclops trees. Although P. acaciicola could be a weak pathogen in a broad range of indigenous plant species, the extremely low disease incidence is an indication of a low level of risk associated with using P. acaciicola as a mycoherbicide. Additionally, pathogenicity trials on indigenous plant species were conducted to give an indication of host susceptibility. A total of 30 indigenous plant species were wound inoculated at two field sites, and potted plants representing 17 indigenous plant species were wound and soil inoculated in a nursery. The optimum growth temperature for P. acaciicola was determined in order to understand it’s seasonal and landscape preference. Mortality was recorded in five of nine indigenous Fabaceae species, while a single plant each of four other non-Fabaceae species died after inoculation. No plants outside the Fabaceae family died in the field. Only A. cyclops seedlings died following soil inoculation. Longitudinal sections of stem inoculated plants revealed no systemic infection in Fabaceae species that survived inoculation. Infection in susceptible Fabaceae species was generally more extensive than infection in susceptible non-Fabaceae species. The optimum growth rate for P. acaciicola was determined at 35°C, indicating an adaptation to summer conditions. Indigenous Fabaceae species do display greater susceptibility than species from other families, indicating some level of specificity, although susceptible species can not be phylogenetically circumscribed. Aside from being a facultative pathogen on A. cyclops, results from this study suggest that P. acaciicola is primarily a saprophyte and an occasional opportunistic pathogen on some indigenous Fabaceae, possibly only being a weak opportunistic pathogen on some non-Fabaceae species. However, the risk of not effectively managing A. cyclops populations in these threatened vegetation types outweighs the risk associated with using P. acaciicola as a mycoherbicide. Therefore the use of P. acaciicola as a mycoherbicide on A. cyclops would be recommended, provided that sufficient monitoring of treated sites is implemented that primarily focus on the indigenous Fabaceae species. The effective control of A. cyclops could be achieved when P. acaciicola is used to compliment current mechanical, biological and chemical control methods in an integrated management strategy.
AFRIKAANSE OPSOMMING: Acacia cyclops, ook bekend as rooikrans, is ‘n indringerplant in Suid-Afrika wat oorspronklik vanaf Australië ingevoer is om die sandduine in die Kaap te stabiliseer. Die verspreiding van rooikrans bedreig die sensitiewe biodiversiteit van die strandveld en kalksteen fynbos. Rooikrans terugsterwing is al vir ‘n geruime tyd opvallend in die grootste deel van die plant se verspreiding in Suid-Afrika, alhoewel die veroorsakende organisme, Pseudolagarobasidium acaciicola, eers onlangs beskryf is. Hierdie swam is as ‘n geskikte kandidaat vir die ontwikkeling van ‘n biologiese onkruiddoder om rooikrans te beheer, genomineer. Alhoewel die huidige biologiese- en meganiese beheer metodes vir rooikrans gedeeltelik suksesvol is, hou dié indringer steeds ‘n ernstige bedreiging vir die natuurlike ekosisteme in. Die gebruik van ‘n swam-gebaseerde onkruiddoder sal die beheer van rooikrans oor die langtermyn meer koste-effektief maak. Die oorgrote meerderheid van die literatuur wat hersien is, ondersteun die gebruik van swam-gebaseerde onkruiddoders as biologiese beheermiddels, veral as die afname in aanvaarbaarheid en beskikbaarheid van chemiese beheermiddels in ag geneem word. Aangesien die Pseudolagarobasidium genus uit saprofiete, opportunistiese fakultatiewe patogene en endofiete bestaan, word daar voorspel dat P. acaciicola ‘n soortgelyke biologiese karakter sal hê. Dit is hoogs waarskynlik dat hierdie swamspesie inheems is, alhoewel die verspreiding wyer mag wees as wat oorspronklik voorspel is. Streng maatreëls moet egter steeds in plek wees om te verseker dat nie-teiken plantspesies nie bedreig word nie. Hierdie studie bestaan uit ‘n unieke risiko-analise met verkeie onderafdelings. ‘n Veld-opname is uitgevoer om die siekte-voorkoms van die inheemse houtagtige plantspesies rondom ‘n 100 siek rooikrans plante te bepaal. DNA ekstraksies is vervolgens vanuit die wortels van siek inheemse plantspesies en -rooikrans uitgevoer, om uiteindelik die teenwoordigheid van P. acaciicola binne die hout te kon bevestig. Uit ‘n totaal van 2432 inheemse houtagtige plante wat aangeteken is, was 22 (0.9%) siek of dood, terwyl die teenwoordigheid van P. acaciicola in 10 van hierdie plante (0.4%), wat ses spesies teenwoordig, bevestig is. Die teewoordigheid van P. acaciicola is ook in 47% van die rooikrans bevestig. Alhoewel P. acaciicola moontlik ‘n swak opportunistiese patogeen op ‘n verskeidenheid inheemse plantspesies is, dui die lae verhouding van dooie inheemse plante teenoor gesonde plante in die veld op ‘n lae risiko vir die gebruik van P. acaciicola as ‘n biologiese onkruiddoder. Patogenisiteitstoetse is op inheemse plantspesies uitgevoer om ‘n aanduiding van gasheervatbaarheid te verkry. Wond-inokulasies is op ‘n totaal van 30 inheemse plantspesies by twee veldstudie-areas uitgevoer, terwyl wond- en grond-inokulasies op 17 inheemse spesies potplante in die kweekhuis uitgevoer is. Die optimale temperatuur waarby P. acaciicola groei, is bepaal om die swam se seisoenale- en habitatsvoorkeure beter te verstaan. Plante van vyf uit die nege inheemse Fabaceae spesies het doodgegaan, terwyl ‘n enkele plant van vier nie-Fabaceae spesies doodgegaan het. Alle plante buite die Fabaceae familie het oorleef in die veld na inokulasie. Slegs rooikranssaailinge het na grond inokulasie doodgegaan. Lengtedeursnee van die stam en wortels van elke geïnokuleerde plant het bevestig dat daar geen sistemiese infeksie in Fabaceae spesies wat inokulasie oorleef het, plaasgevind het nie. Infeksies in vatbare Fabaceae spesies was oor die algemeen meer ernstig as infeksies in vatbare nie-Fabaceae spesies. Die optimale groei van P. acaciicola het by 35°C plaasgevind, wat aandui op ‘n voorkeur vir somerstoestande. Inheemse Fabaceae spesies het meer vatbaar as vatbare plantspesies van ander families voorgekom. Hierdie verskynsel dui op ‘n sekere vlak van spesifisiteit, alhoewel daar geen duidelike filogenetiese grense vir vatbare spesies bepaal kon word nie. Behalwe vir die feit dat P. acaciicola ‘n fakultatiewe patogeen op rooikrans is, stel resultate van hierdie studie voor dat hierdie swam hoofsaaklik ‘n saprofiet is wat soms ook ‘n opportunisties patogeen op sekere inheemse Fabaceae is en moontlik slegs ‘n swak opportunistiese patogeen op plantspesies buite die Fabaceae familie is. Die swak en oneffektiewe bestuur van rooikrans in hierdie bedreigde plantegroeitipes hou egter ‘n groter bedreiging in as die gebruik van P. acaciicola as ‘n biologiese onkruiddoder. Pseudolagarobasidium acaciicola word daarom aanbeveel vir die beheer van rooikrans, mits voldoende monitering, wat fokus op inheemse Fabaceae spesies, gepaard gaan met die gebruik van hierdie biologiese onkruiddoder. Rooikrans kan effektief beheer word as P. acaciicola ingespan word om huidige meganiese-, biologiese- en chemiese beheermetodes in ‘n geïntegreerde bestuurstrategie te komplimenteer.

Chenopodium album is considered one of the most important weeds adversely affecting agricultural production due to its highly competitive influence on field crops. Chemical herbicides have increased the efficiency of farming, but recently problems of herbicideresistant weed populations and herbicide residues in soil, water, food products and effects on non-target organisms have increased, consequently, other methods of control of weeds by using specific fungi as herbicides have been suggested. The purpose of this research was to evaluate the biological control of the weed Chenopodium album by the fungus Ascochyta caulina. Some of the factors which control dormancy and germination of Chenopodium album seeds have been investigated to understand better the weed population dynamics. The results showed that seeds from two populations (UK and Libya) differ in their response to factors such as light, chilling, and burying in soil. This could have implications for effective control of the weed in different regions. Two formulations of mycoherbicides (Tween 80 and Gelatine based applications) were tested in the laboratory, and showed promise in reducing growth of the weed, especially the formula of Tween 80. There was extensive shoot fresh and dry weight reduction of inoculated Chenopodium album, as well as reduced root growth. Highest disease severity rates were observed on plants in the first three week of life. A field trial revealed similar results but less disease severity was observed, possibly because of dry weather. However, it was concluded that the fungus Ascochyta caulina is a potentially useful biological control agent but many factors still can be modified in relation to application of the mycoherbicide to increase its efficacy.
Libyan Government

Chenopodium album is considered one of the most important weeds adversely affecting agricultural production due to its highly competitive influence on field crops. Chemical herbicides have increased the efficiency of farming, but recently problems of herbicideresistant weed populations and herbicide residues in soil, water, food products and effects on non-target organisms have increased, consequently, other methods of control of weeds by using specific fungi as herbicides have been suggested. The purpose of this research was to evaluate the biological control of the weed Chenopodium album by the fungus Ascochyta caulina. Some of the factors which control dormancy and germination of Chenopodium album seeds have been investigated to understand better the weed population dynamics. The results showed that seeds from two populations (UK and Libya) differ in their response to factors such as light, chilling, and burying in soil. This could have implications for effective control of the weed in different regions. Two formulations of mycoherbicides (Tween 80 and Gelatine based applications) were tested in the laboratory, and showed promise in reducing growth of the weed, especially the formula of Tween 80. There was extensive shoot fresh and dry weight reduction of inoculated Chenopodium album, as well as reduced root growth. Highest disease severity rates were observed on plants in the first three week of life. A field trial revealed similar results but less disease severity was observed, possibly because of dry weather. However, it was concluded that the fungus Ascochyta caulina is a potentially useful biological control agent but many factors still can be modified in relation to application of the mycoherbicide to increase its efficacy.

Bromus tectorum and other annual brome grasses have invaded many ecosystems of the western United States, and because of an annual-grass influenced alteration of the natural fire cycle on arid western range lands near monocultures are created and conditions in which the native vegetation cannot compete are established. Each year thousands of hectares become near monocultures of annual brome grasses. Pyrenophora semeniperda, a generalist seed pathogen of annual grasses, shows major potential as a possible mycoherbicide that could help in reducing the monocultures created by annual grasses. The purpose of this research was to identify the requirements for isolating cultures of P. semeniperda, search for a hypervirulent strain, and evaluate its effect in the field. The techniques for isolating the fungus have evolved and become more efficient. The first two years of working with P. semeniperda resulted in 11 isolates. During the third year of this study, we developed a single spore isolation technique that resulted in 480 additional isolates. Virulence screening resulted in detection of a range of isolate ability to kill non-dormant B. tectorum seeds. Ninety-two isolates represented a range of virulence from 0-44%. The variation in virulence was expressed mostly within populations rather than between populations. Similarly, virulence varied significantly within Internal Transcribed Spacer (ITS) genotypes and habitats but not between them. When conidial inoculum was applied in the field there was no observed difference in disease incidence between different levels of inoculum. This is thought to have been due to applying the inoculum under conditions in which most in situ seeds were infected and killed by already high field inoculum loads. While additional field trials are needed to optimize the inoculum effectiveness, the overall results of this research provide a good foundation for using P. semeniperda as a biological control for seed banks of annual brome grasses.

Studies were carried out to assess the potential of the plant pathogenic fungus Ascochyta caulina as a mycoherbicide against Chenopodium album L., a worldwide weed in arable crops. A. caulina is associated with necrosis of leaves and stems of plants belonging to genera of Chenopodium L. and Atriplex L. The research of this thesis was focused on recognition of factors that may enhance pathogenicity and determine the impact of the pathogen not only on its target host (growing both alone and in competition), but also on a range of temperate crop species. These studies showed that formulation of A. caulina spores with nutrients, especially V-8, and surfactants resulted in faster and greater in vitro germination. During an investigation of in vitro pathogen growth, it was observed that A. caulina grows most prolifically in the nutrient rich liquid medium, V-8. Simple nitrogen and carbon sources also induce a positive effect on the pathogen's growth. Nutrients also promoted the pathogen's enzyme activity of a-amylase and cellulase. The in vitro and in planta enzyme activity varied significantly between A. caulina isolates and plant growth stage affected the a-amylase activity but not cellulase. Observations of A. caulina on detached leaves and whole plants of C. album and several crop species, showed that the surface deposits on C. album leaves are part of a defensive mechanism, and that the pathogen can develop pycnidia on the dead leaves of the non-host plants. A. caulina caused no infection symptoms on any of the crop species. However, under some circumstances it reduced the dry biomass accumulation of some of the non-target plants. Results of the three-way interaction between Beta vulgaris, C. album and the potential mycoherbicide A. caulina showed that the pathogen can reduce the competitive effects of the weed over B. vulgaris by suppressing the growth of the former.

The overall objective of this study was to test the hypothesis that insects can vector F. tumidum conidia to infect gorse plants with the aim of developing an alternative approach to mycoherbicide delivery to control weeds. Four potential insect species (Apion ulicis, Cydia ulicetana, Epiphyas postvittana and Sericothrips staphylinus) were assessed for their ability to vector F. tumidum conidia. To achieve this, the external microflora (bacteria and fungi) and the size and location of fungal spores on the cuticle of these insect species were determined. In addition, the ability of the insects to pick up and deposit F. tumidum conidia on agar was studied. Based on the results from these experiments, E. postvittana was selected for more detailed experiments to determine transmission of F. tumidum to infect potted gorse plants. The factors promoting pathogenicity of F. tumidum against gorse and the pathogen loading required to infect and kill the weed were also determined. The external microflora of the four insect species were recovered by washing and plating techniques and identified by morphology and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and sequencing of internally transcribed spacer (ITS) and 16S rDNA. A culture-independent technique (direct PCR) was also used to assess fungal diversity by direct amplification of ITS sequences from the washings of the insects. All insect species carried Alternaria, Cladosporium, Nectria, Penicillium, Phoma, Pseudozyma spp. and entomopathogens. Ninety four per cent of the 178 cloned amplicons had ITS sequences similarity to Nectria mauritiicola. E. postvittana carried the largest fungal spores (mean surface area of 125.9 ìm2) and the most fungal CFU/insect. About 70% of the fungi isolated from the insects were also present on the host plant (gorse) and the understorey grass. The mean size of fungal spores recovered from the insect species correlated strongly with their body length (R² = 85%). Methylobacterium aquaticum and Pseudomonas lutea were common on all four insect species. Pseudomonas fluorescens was the most abundant bacterial species. In the pathogenicity trials, the effectiveness of F. tumidum in reducing root and shoot biomass of 16 and 8 wk old gorse plants was significantly increased with wounding of the plants. Older plants (32 wk old) which were wounded and inoculated were significantly shorter, more infected and developed more tip dieback (80%) than plants which were not wounded (32%). This indicates that damage caused by phytophagous insect species present on gorse through feeding and oviposition may enhance infection by F. tumidum. Wounding may release nutrients (e.g. Mg and Zn) essential for conidia germination and germ tube elongation and also provide easier access for germ tube penetration. Conidial germination and germ tube length were increased by 50 and 877%, respectively when incubated in 0.2% of gorse extract solution for 24 h compared with incubation in water. Inoculum suspensions amended with 0.2% of gorse extract caused more infection and significantly reduced biomass production of 24 wk old gorse plants than suspensions without gorse extract. A minimum number of about 900 viable conidia/infection site of F. tumidum were required to infect gorse leaves. However, incorporation of amendments (which can injure the leaf cuticle) or provision of nutrients (i.e. gorse extract or glucose) in the formulation might decrease the number of conidia required for lesion formation. Scanning electron micrographs showed that germ tube penetration of gorse tissue was limited to open stomata which partly explain the large number of conidia required for infection. The flowers and leaves were more susceptible to F. tumidum infection than the spines, stems and pods. An experiment to determine the number of infection sites required to cause plant mortality showed that the entire plant needs to be inoculated in order for the pathogen to kill 10 wk old plants as F. tumidum is a non systemic pathogen. The number of infection sites correlated strongly with disease severity (R² = 99.3%). At least 50% of the plant was required to be inoculated to cause a significant reduction in shoot dry weight. F. tumidum, applied as soil inoculant using inoculated wheat grains in three separate experiments, significantly suppressed gorse seedling emergence and biomass production. In experiments to determine the loading capacity of the insect species, E. postvittana, the largest insect species studied, carried significantly more (68) and deposited significantly more (29) F. tumidum conidia than the other species. Each E. postvittana, loaded with 5,000 conidia of F. tumidum, transmitted approximately 310 conidia onto gorse plants but this did not cause any infection or affect plant growth as determined by shoot fresh weight and shoot height. E. postvittana on its own did not cause any significant damage to gorse and did not enhance F. tumidum infection. It also failed to spread the pathogen from infected plants to the healthy ones. There was no evidence of synergism between the two agents and damage caused by the combination of both E. postvittana and F. tumidum was equivalent to that caused by F. tumidum alone. This study has shown that E. postvittana has the greatest capacity to vector F. tumidum since it naturally carried the largest and the most fungal spores (429 CFU/insect). Moreover, it naturally carried Fusarium spp. such as F. lateritium, F. tricinctum and Gibberella pulicaris (anamorph Fusarium sambucinum) and was capable of carrying and depositing most F. tumidum conidia on agar. Coupled with the availability of pheromone for attracting the male insects, E. postvittana may be a suitable insect vector for delivering F. tumidum conidia on gorse using this novel biocontrol strategy. Although it is a polyphagous insect, and may visit non-target plants, F. tumidum is a very specific pathogen of gorse, broom and a few closely related plant species. Hence, using this insect species to vector F. tumidum in a biological control programme, should not pose a significant threat to plants of economic importance. However, successful control of gorse using this "lure-load-infect" concept would depend, to a large extent on the virulence of the pathogen as insects, due to the large size of F. tumidum macroconidia, can carry only a small number of it.

Development of microbial herbicides is constrained by unreliability in the field where conditions are often sub-optimal for infection. Crucially, sufficient moisture, often dew, is required to establish infection. Two model systems, Colletotrichum dematium (Pers. ex Fr. ) Grove on Chenopodium album L. and Mycocentrospora acerina (Hartig) Deighton on Viola arvensis Murr., have been investigated and formulation requirements for each system identified, principally to reduce the dew period necessary for infection. Effects of adjuvants (surfactants, stickers and humectants) on spore germination and appressorium formation were investigated in vitro. Few were toxic and then, principally, at high concentration. The surfactants Tween 40,60 and 80 were compatible with both pathogens. Similarly, the stickers acacia, ghatti, guar, karaya, locust bean and xanthan gums and low viscosity alginic acid were all non-toxic as was the humectant glycerol. Each pathogen reacted differently to the adjuvants and any potential microbial herbicide will need individual matching of adjuvants to give an effective formulation. A working formulation (rapeseed oil-in-water (1: 10 v/v) emulsion using 0.1 % v/v Tween 40 as the emulsifier) was found to reduce the dew period requirement of M. acerina from 36 to 18 hours. The formulation protected spores from desiccation for 24 hours after application, or for 16 hours following a sub-optimum dew period occurring immediately after application. Scanning electron microscopy showed that the applied spores, and the developing mycelium, were immersed in the oil deposit. Transmission electron microscopy of sections through formulation deposits on the leaf revealed that some inversion of the emulsion, to form a water-inoil deposit, had occurred, suggesting a mechanism of protection against desiccation. The oil phase infiltrated the cortical intercellular spaces only when the leaf was infected. This intercellular oil contained more water than that on the leaf surface. Emulsion-formulation applied to run-off with an 'air brush', consistently gave significantly better weed control under sub-optimal dew conditions than a formulation of surfactant only. When applied with a conventional hydraulic nozzle at 400 1 ha" the emulsion was only occasionally superior to the surfactant alone. Such interactions require further in-depth investigation. The importance of correct inoculum placement for maximum effectiveness, independent of formulation type, was highlighted. Unless all meristems are killed, survivors quickly grow, despite the death of neighbouring leaves and petioles, and the weed suffers merely a growth check. Formulation as emulsion improved diseasee stablishmenta nd diseasee xpressioni n the target weedo nly in somec ircumstancesF. urther researchin to spraya pplicationm ethodsa ndt heir interactions with formulation, host and environment is clearly necessary

Un projet visant à identifier des mycoherbicides pour lutter contre les adventices a été initié entre l’UMR Agroécologie de Dijon et la société DE SANGOSSE® (Agen). Trois volets ont structuré ce projet à l’issue d’une collecte de prélèvement de 475 plantes représentatives de 23 espèces d’adventices symptomatiques et asymptomatiques en Bourgogne et en Beauce. Le 1er volet reposait sur une approche de type metabarcoding (technologie Illumina), pour évaluer et comparer la diversité des communautés fongiques endophytes des plantes symptomatiques et asymptomatiques. 542 genres fongiques ont ainsi été identifiés. Des taxons associés aux plantes symptomatiques ont été identifiés. Parmi ceux-ci, certains sont des pathogènes connus, d’autres non et ils constituent des pistes à exploiter pour la recherche de candidats mycoherbicides. Le deuxième volet repose sur une approche conventionnelle de microbiologie et pathologie. Une collection de 194 champignons associés aux symptômes des adventices a été constituée. La pathogénicité de ces isolats a été testée grâce à une série de screenings de plus en plus sélectifs qui ont abouti à la sélection de cinq souches, identifiées par séquençage de l’ITS ou d’autres marqueurs taxonomiques. Une souche appartient à l’espèce Boeremia exigua var exigua, une autre à l’espèce Alternaria alternata, deux appartiennent à l’espèce A. penicillata et la dernière au genre Alternaria. Le troisième volet visait à identifier le mode d’action d’une souche par une double approche, métabolomique et microscopique. La souche de B. exigua var exigua secrète des métabolites phytotoxiques mais également infeste et semble détruire les tissus végétaux sous-épidermique de la plante hôte.Ce projet exploratoire a fourni des pistes de taxons fongiques associés à des symptômes observés sur adventices en analysant la diversité par une approche moléculaire et a fourni des souches fongiques, mycoherbicides potentiels, par une approche microbiologique dont on voit bien qu’elle reste une méthode incontournable, malgré ses limites, pour obtenir des candidats fongiques à action herbicide
A project aiming at identifying mycoherbicides to control weeds has been initiated between the UMR Agroécologie (Dijon) and the company DE SANGOSSE® (Agen, France). Three axes structured this project after a sampling collection of 475 plants representative of 23 species of symptomatic and asymptomatic weeds was carried out in Burgundy and Beauce. The first part was based on a metabarcoding approach (Illumina technology), to evaluate end compare the diversity of endophytic fungi communities of symptomatic and asymptomatic weeds. 542 fungal genera have been identified. Taxa associated with symptomatic plants have been identified. Of these, some are known pathogens, others are not, and both constitute avenues to exploit for the research of mycoherbicide candidates. The second axe is based on a conventional approach to microbiology and pathology. A collection of 194 fungi associated with weed symptoms was established. The pathogenicity of these isolates was tested through a series of increasingly selective screenings that resulted in the selection of five strains that were identified by sequencing of ITS or other taxonomic markers. One strain belongs to the species Boeremia exigua var exigua, another species Alternaria alternata, two belong to the species A. penicillata and the last to the genus Alternaria. The third axe aimed at identifying the mode of action of a strain by a dual metabolomics and microscopic approach. The strain of B. exigua var exigua produced phytotoxic secondary metabolites but also infested and apparently destroyed the sub-epidermal plant tissues of the host plant.This exploratory project provided tracks to exploit fungal taxa associated with observed weeds symptoms, by analyzing the diversity, by a molecular approach and provided fungal strains, potential mycoherbicides by a conventional microbiological approach that we can see it remains an unavoidable method, despite its limitations, to obtain fungal candidates with herbicidal action