Dissertations / Theses on the topic 'Phytophthora cinnamomi control'

Phytophthora cinnamomi Rands is a common and devastating pathogen of cultivated proteas worldwide. Webb (1997) described a Sudden Death plant disease of proteas in Western Australia (WA) protea plantations. Proteas that suffer the syndrome display symptoms such as stunted growth, wilting, chlorosis and often death. In the current study, a number of protea plantations in the southwest of WA were visited to quantify the extent that P. cinnamomi was attributing to deaths of cultivated proteas. The survey indicated that P. cinnamomi is the major cause of Sudden Death in proteas. A range of other fungi (Fusarium, Botryosphaeria, Pestalotiopsis, Alternaria) and pests (nematodes, mealy bug, scale insects) were also identified to be contributing to protea death and decline in WA plantations. In many cases the factors contributing to protea disease appeared complex, with a range of physical factors or nutritional imbalances commonly associated with these pathogens and pests. As P. cinnamomi was the major cause of death of cultivated proteas the remainder of the experiments described in this dissertation investigated its control in horticultural plantings. Biofumigation has the potential to become an important technique in an overall integrated management approach to P. cinnamomi. In this thesis, biofumigation refers to the suppression of pathogens and pests by the incorporation of Brassica plants into the soil. Two biofumigants (Brassica juncea (L.) Czern., B. napus L.) were screened for their effect on the in vitro growth of five common Phytophthora species (P. cinnamomi, P. cactorum (Lebert & Colin) Schroeter., P. citricola Sawada, P. cryptogea Pethyb. & Laff. and P. megasperma Drechsler). Growth was determined by the measuring dry weight and radial growth of vegetative hyphae. B. juncea was found to be superior in its suppressive effect compared to B. napus. There was also significant variation in the sensitivity of the Phytophthora species to the suppressive effects of the biofumigants. P. cinnamomi was the most sensitive of the five species investigated. Where the rates of the biofumigant were sufficient to suppress growth of Phytophthora, the suppressive effect was mostly fungicidal. To determine how B. juncea and B. napus affect the infective ability and survival of P. cinnamomi, their effects on sporangia and chlamydospores production in soil was investigated in vitro. P. cinnamomi colonised Miracloth discs were added to soil amended with the two Brassica species, before being removed every two days over an eight day period for the determination of sporangia production, chlamydospore production and infective ability. Only the soils amended with B. juncea significantly reduced sporangia production in P. cinnamomi. Both Brassica species increased the percentage of aborted or immature sporangia and reduced the infective ability of the pathogen. Neither Brassica species had any effect on zoospore release or chlamydospore production in P. cinnamomi. Soil cores and soil leachate were collected from biofumigant-amended field soils to determine the inoculum potential and infective ability of the pathogen under glasshouse conditions. Amending the soil with both Brassica species had an immediate suppressive effect on the inoculum potential and infective ability of the P. cinnamomi. However, after this initial suppression there was a gradual increase in the recovery of the pathogen over the monitoring period of four weeks. To determine if the suppression would result in decreased disease incidence in a susceptible host, Lupinus angustifolius L. seeds were planted in the biofumigant amended soil. B. juncea amended soils reduced the disease incidence of P. cinnamomi by 25%. B. napus had no effect on disease incidence in L. angustifolius. Although the current study had demonstrated that biofumigants could suppress the growth, sporulation and infection of P. cinnamomi, it was unclear if this would equate to a reduction in disease incidence when applied in the field. A field trial was conducted on a protea plantation in the southwest of Western Australia that compared biofumigation with B. juncea to chemical fumigation (metham sodium) and soil solarisation. The three soil treatments were used in an integrated management approach to control P. cinnamomi that included the use of a hardwood compost, mulch and water sterilisation. All treatments were monitored during their application to ensure the treatments were conducted successfully. The three soil treatments significantly reduced the recovery of the pathogen and the infective ability of the pathogen to a soil depth of 20 cm. Metham sodium was the most suppressive soil treatment and soil solarisation was the least suppressive treatment. Only the metham sodium treatment resulted in a significant reduction in the incidence of root rot in Leucadendron salignum P.J. Bergius x laureolum (Lam.) Fourc (c.v. Safari Sunset) over the monitoring period of three years. Another field trial was conducted on the same protea plantation to compare the effectiveness of B. juncea and B. napus, without the use of other control strategies, to reduce the incidence of P. cinnamomi infection of Leucadendron Safari Sunset. The concentration of isothiocyanates was monitored for seven days after the incorporation of the biofumigants. Although both Brassica species reduced the recovery and infective ability of the pathogen, neither biofumigant reduced the incidence of root rot in Leucadendron Safari Sunset. In conclusion, P. cinnamomi is the most common and devastating pathogen in WA protea plantations. The current study demonstrated that P. cinnamomi is sensitive to the suppressive nature of biofumigants. Biofumigants can suppress the in vitro growth, sporulation, infective ability of P. cinnamomi and reduce the incidence of the disease caused by the pathogen in the glasshouse. Of the two Brassica species investigated, B. juncea was superior in its ability to control P. cinnamomi compared to B. napus. When applied in the field, biofumigation using B. juncea was found to be more suppressive that soil solarisation, but not as effective as metham sodium.

One of the major aims of the research presented in this thesis was to assist managers of native vegetation communities in southeastern Australia in understanding the dynamics of P. cinnamomi with an important ecological species, Xanthorrhoea australis. It trialed the use of phosphite in large-scale field applications to establish the usefulness of this management option for the first time on Victorian flora. This thesis describes the process of disease development within mature X. Australia plants. For the first time it was shown that within X. australis plants, secondary disease symptoms are related to the percentage of stem that has been infested by the disease. It was evident that after initial invasion the pathogen moves via root xylem and throughout the plant within vascular to the stem, especially within the desmium. The research shows that the pathogen could not be isolated consistently even though it was considered to be responsible for disease symptoms. Trials of a control fungicide (Foli-R-fos 200) shows that protection occurs in many susceptible plants when 2 and 6g a.i./L phosphite is applied. Phytotoxicity occurred in native plants at Anglesea and within controlled environment trials when using ≥ 6g a.i./L. It will be shown that 2g a.i./L phosphite controls disease in sprayed plots within heathlands at Anglesea and a recently burnt coastal woodland community at Wilson’s Promontory. The proportion of healthy X. australis plants treated with phosphite was significantly higher than the proportion in control plots without phosphite. The research shows that phosphite was recovered from leaves of three species treated with Foli-R-fos 200 in the field. For the first time it has been shown that seed germination was reduced in two species when high concentrations of phosphite were applied. The first documentation of the effect that phosphite has on soil properties showed that nitrogen and oxidised organic carbon were the only parameters to alter significantly. This thesis provides answers to some important questions, answers that can now be used by managers in formulating better policies and actions at an operational level. There has been a dire need in Victoria to address many issues regarding P. cinnamomi and this thesis provides relevant and informative approaches to disease control, and a better understanding of the disease progress.

The overall aim of this project was to screen actinomycetes for the ability to produce antibiotics inhibitory to P. cinnamomi in vitro. Some of the more promising isolates were then screened in vivo in a glasshouse pot-trial. The actinomycete isolates used had been previously isolated from jarrah forest soils suppressive to P. cinnamomi, ie. areas where P. cinnamomi was present in the soil but there was little evidence of disease. They had been isolated using dry heat pretreatments ( 120°C for one hour) designed to select for less frequently isolated actinomycetes especially the genus Microbispora. Isolates were screened against P. cinnamomi in vitro for the production of secondary metabolites inhibitory to this pathogen. Of the isolates screened 40% caused strong inhibition of P. cinnamomi , 13% caused medium inhibition and 46% caused no inhibition of P. cinnamomi. Of the 40% of isolates that caused inhibition in vitro only ±4.0% of those tested for inhibition of P. cinnamomi in the pot-trial showed a potential for inhibiting P. cinnamomi in soil. The pot-trial was performed using actinomycete isolates that had shown different levels of inhibition in vitro. Eight actinomycetes that had produced an antibiotic inhibitory to P. cinnamomi in vitro were tested in the pot-trial, 6 showing strong inhibition and 2 medium inhibition, but only 2 of these isolates a Streptomyces, isolate 97, and a maduromycete, isolate 55A, suppressed P. cinnamomi in vivo. In addition 2 of the 4 isolates with no in vitro inhibition of P. cinnamomi showed some activity against P. cinnamomi in the pot-trial. The results emphasised the fact that the production of an antibiotic in vitro does not automatically mean that the isolate will have the ability to control the pathogen in vivo. The actinomycete isolates 97 and 55A which had shown strong inhibition in the in vitro screening were also screened against a range of fungal plant pathogens from different taxonomic groups. The fungal pathogens included representatives from the ascomycetes, hyphomycetes, basidiomyeetes and oomycetes. Eight other species of Phytophthora were also included. Isolate 97 showed the ability to strongly inhibit all of the fungal pathogens except Pythium ultimum which was only partially inhibited. Isolate 55A did not have the ability to inhibit Pythium ultimum but did inhibit all other pathogens to different degrees. Both isolates though showed that the antibiotics they produced were active over a range of different fungal taxonomy groups. The metabolites from Streptomyces isolate 97 which showed strong activity against a range of fungal pathogens were extracted and purified from a broth culture filtrate. The antibiotic compound did not lose its ability to inhibit P cinnamomi through the extraction or purification process. Two antibiotic compounds were extracted, YB1 and YB2, both of which were inhibitory to P cinnamomi although YB2 was the stronger of the two. Antibiotics YBI and YB2 were also inhibitory to a few bacterial plant pathogens increasing the range of pathogens that could be inhibited by isolate 97. Due to the wide range of pathogens inhibited by Streptomyces isolate 97 and its metabolites an attempt was made to further identify the isolate to species. There was not sufficient time to determine all the data required to identify the isolate by numerical classification, which requires data from 139 unit characters but a simple outdated key was used in the attempt. The use of the older key did not result in the identification of the isolate but the isolate was characterised morphologically and with respect to carbon source utilisation.

ABSTRACT The plant pathogen Phytophthora cinnamomi is having a major negative impact on the biodiversity of native ecosystems in the south west of Western Australia. Acacia pu/chella has previously been shown to suppress P. cinnamomi in the jarrah forest of Western Australia and protect susceptible species from infection. This has management implications for the control of P. cinnamomi in rehabilitated bauxite pits infested with the pathogen and severely diseased forest areas. The objective of this thesis was to determine if other Western Australian native legume species have the potential to biologically control P. cinnamomi. In a rehabilitated bauxite pit trial, five Acacia species were planted with Banksia grandis, to determine their ability to protect this highly susceptible species against P. cinnamomi infection. A. pulchella protected B. grandis from infection for over a year. This protection was not the result of a decrease in soil moisture or soil temperature, as was previously suggested. A. urophylla, A. extensa, A. latericola and A. drummondii did not protect B. grandis in this trial. The trial was replicated in the glasshouse under conditions conducive to the pathogen. The infection of B. grandis by P. cinnamomi was delayed for up to 7 weeks by all of these Acacia species; however, none of them protected B. grandis from eventual mortality. In a glasshouse soil inoculation trial, native legumes other than A. pulchella were able to reduce the soil inoculum potential of P. cinnamomi. Based on these findings, the species with the greatest potential for biological control of P. cinnamomi along with A. pulchella were A. extensa, A. stenoptera and A. a/ata. 11 By assessing the roots of soil inoculated native legumes from the glasshouse trial, P. cinnamomi was found to asymptomatically infect fine lateral roots of some species and sporulate from them. These species can potentially harbour the pathogen and allow for an inoculum increase when environmental conditions are favourable. A. urophylla and Viminaria juncea were the species with the least potential for biological control of P. cinnamomi due to this finding. A possible management tool for bauxite pits in infested areas and in severely diseased forest areas is the ability to influence the density and composition of species used for rehabilitation, by manipulating the seed mix ratio. The implications of this study would be to increase seed in a seed mix of those legume species with the potential for biological control and decrease seed of those species that can harbour the pathogen. However, before rehabilitation management practices are adjusted, further investigation is required to understand how P. cinnamomi suppression occurs and whether it is transferable to a natural environment. The actions of P. cinnamomi suppression by legume species and future research directions are discussed.

The soil borne plant pathogen Phytophthora cinnamomi has irreversibly altered the make-up and diversity of the plant communities found in Australia. Recently, the fungicide phosphite has been used to effectively reduce the impact of this pathogen in natural plant communities. However, little is known (a) about how rapidly phosphite is diluted in the tissues of rapidly growing plants and (b) how soil and plant phosphate levels interact with phosphite and its ability to induce host-resistant responses when challenged by P. cinnamomi. This study examined the effects of different phosphite rates (0, 24 and 48 kg/ha phosphite) applied as a mist application on three size classes of Banksia grand is, as well as the interaction of phosphate status on two Eucalyptus marginata forest vegetation types differing in soil phosphate status with phosphite. It also examined, under controlled glasshouse conditions, the effects different soil phosphate levels had on in planta phosphite and phosphate levels in B. hookeriana, and the subsequent control of P. cinnamomi. This study was the first to look at the role of phosphate in the soil and the plant, and its interaction with phosphite and the subsequent control of P. cinnamomi in planta. Results from the field trial indicated that phosphate in the soil did not play a role in the reducing the uptake of phosphite by the plant. It did suggest that stem and root colonisation was increased when phosphate in the soil was more plentiful. Further research is needed into this area. This study was also the first to look at the distribution of phosphite in planta. The highest concentration of phosphite was in the leaves, followed by the stem and then roots. Ph9sphite in the plant tissue was found to increase as the phosphite applied to 5 the plants increased. Plants classed as seedlings showed more phytotoxic symptoms than the intermediate and semi-mature plants. The concentration of phosphite in the roots of the intermediate sized plants was more than double the amount found in the seedling and semi-mature plants. The concentration of phosphite in the whole plant, as well as in the leaves and stems per plant, increased as the plant size increased. This was supported by results that showed that as the dry weight of the leaves increased so did the amount of phosphite in the leaves. The same was seen with the dry weights of the stems and roots that correlated with phosphite in the stem and the roots, respectively. Lesions and P. cinnamomi colonisation in the stems of non-phosphite treated plants were more than double those in stems of plants treated with 24 and 48 kg/ha phosphite. There was very little difference in the visible lesion lengths and P. cinnamomi colonisation between plants treated with 24 and 48 kglha of phosphite even though plants sprayed with 48 kg/ha phosphite had significantly more phosphite in their tissues than plants sprayed with 24kg/ha phosphite. This suggests that the phosphite in the plant may have been metabolised into another substance and that this substance was acting on the pathogen and/or the plant to reduce colonisation. This was further supported by no observed correlation between phosphite in the plant tissue and the extent of colonisation or visible stem lesion caused by P. cinnamomi. This was contradictory to other results in this study (Chapter 2) that clearly showed that phosphite did restrict the colonisation of the pathogen. Further research is needed into the mode of action of phosphite. In the glasshouse trial, a non-invasive inoculation technique failed to infect B. hookeriana plants with the pathogen. However, this is likely due to very high ambient temperatures experienced during the trial, since a preliminary trial 18 days earlier resulted in extensive colonisation of all plants inoculated. As phosphate levels increased, stem colonisation by the pathogen increased in the presence of phosphite. There was no difference in the concentration of phosphite in the leaves. As phosphite applied increased, so did the concentration of phosphite in the root tissue. This study shows that phosphate does interact with phosphite and the subsequent expression of P. cinnamomi, and as phosphate levels increased in planta so did the extent of colonisation by the pathogen. The exact nature of this interaction is still unknown and further research is required to better understand the nature of this relationship.

Mestrado em Engenharia Agronómica - Protecção das Plantas - Instituto Superior de Agronomia - UL
O declínio dos Montados de sobro e azinho é uma doença que tem sido descrita desde a década de 80 do século passado e é influenciada pela interacção de factores bióticos e abióticos. Inúmeros estudos mostram uma associação entre espécies do género Phytophthora e o declínio dos Montados, sendo que Phytophthora cinnamomi é a espécie isolada com mais frequência nos solos desses ecossistemas. P. cinnamomi é um patogénio do solo, da classe Oomycota, que causa podridão radicular e, consequentemente, a morte da planta. A sua erradicação dos solos é muito difícil. Os produtos fitofarmacêuticos utilizados até ao momento não apresentam eficácia no seu controlo, pois o patogénio encontra-se disseminado nos solos e apresenta uma elevada gama de hospedeiros. Tais condicões favorecem a sua sobrevivência. Dado que a luta química para o controlo de P. cinnamomi tem-se mostrado ineficaz, é necessário procurar alternativas mais sustentáveis. Este trabalho teve como objectivo apresentar um primeiro contributo para a selecção de plantas com um efeito alelopático sobre P. cinnamomi. Para tal, seleccionaram-se doze espécies das seguintes famílias: Fabaceae, Poaceae, Lamiaceae e Brassicaceae. Avaliou-se a susceptibilidade das espécies selecionadas a P. cinnamomi em ensaios em estufa, tendo-se observado que três espécies foram infectadas. Realizaram-se ensaios in vitro de modo a testar os extractos aquosos radiculares das plantas e determinar os seus efeitos na actividade do patogénio através do seu crescimento micelial, produção de esporângios e clamidósporos bem como da viabilidade e germinação de zoósporos. Como resultado, seleccionou-se as espécies Eruca sativa e Raphanus raphanistrum por inibirem quase totalmente o crescimento e desenvolvimento do patogénio, O efeito inibitório total fna actividade do patogénio foi observado no extracto combinado das duas espécies. Por outro lado, os extractos de gramíneas, em particular de Lolium rigidum, tiveram um efeito promotorr do patogénio
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Uma característica notável da interacção entre plantas e microrganismos patogénios de espécies de Phytophthora é a produção de proteínas inibidoras de glucanases (GIP) relacionadas com a doença da Tinta do castanheiro. Dada a grande importância do castanheiro (Castanea sativa Mill) ao nível da economia e ecologia na região do Nordeste Transmontano, tornou-se necessário melhorar o conhecimento sobre os mecanismos de infecção de Phytophthora cinnamomi Rands, através do estudo da proteína GIP como mecanismo de resposta a proteínas hidrolíticas, endo-β-1,3-glucanases por parte da planta. Este estudo, teve como objectivo clonar o gene gip e avaliar a expressão por gel de SDS-PAGE em diferentes tempos de indução e determinar em qual dos substratos naturais utilizados existe maior expressão por RT-qPCR. Paralelamente foram efectuados biotestes com plantas Castanea sativa Mill e com Phytophthora cinnamomi de modo a estudar a capacidade antimicrobiana de óleos essenciais extraídos de Mentha pulegium L. Os resultados deste estudo revelaram que a clonagem foi bem sucedida após a visualização em gel de agarose 0.8 % (v/v) de uma banda de 5369pb e outra de 940pb corresponde ao vector pET-28a(+) e á ORF do gene gip. A expressão da proteína verificou-se às 8 horas de indução pela presença de uma banda 31kDa observada por gel SDS-PAGE. A análise da expressão por RT-qPCR indicou que a expressão do gene gip é maior quando o patogénio cresce na presença de serrim 0.2 % (p/v) como substrato indutor. Os ensaios com óleos essenciais extraídos de Mentha pulegium L revelaram que a P. cinnamomi é inibida a uma concentração de 80 % (v/v), na qual a planta se mantem viavel e a sua sobrevivencia não é afectada. Desta forma o uso deste produto natural como agente activo é de grande importância, especialmente nas regiões que têm soutos como recursos naturais, de grande valor económico, podendo vir a ser uma alternativa ao controlo de P. cinnamomi.
A remarkable characteristic of the interaction between plants and pathogen microorganisms of Phytophthora species is the production of inhibitory proteins of glucanases (GIP) related with the chestnut ink disease. Due to the great importance of the chestnut (Castanea sativa Mill) at economical and ecological levels in the Nordeste Transmontano region, became necessary to improve the knowledge about the infection mechanisms of Phytophthora cinnamomi Rands, through the study of GIP proteins, as a response mechanism to hydrolytic proteins, endo-β-1,3-glucanases, by the plant. In the present study was intended to clone the gene gip and to evaluate the expression by SDS-PAGE gel in different induction times and to determine in which natural substrates used there is a higher expression for RT-qPCR. At the same time were carried out bioassays with Castanea sativa Mill plants and Phytophthora cinnamomi in order to study the antimicrobial activity of the essential oils extracted from Mentha pulegium L. The results obtained revealed that the cloning was well succeeded after the visualization in a agarose 0.8 % (v/v) gel of two bands of 5369pb and 940pb corresponding respectively to the vector pET-28a(+) and to the ORF of the gip gene. The protein expression was observed at 8 hours of induction by the presence of a band of 31kDa in a SDS-PAGE gel. The analysis of expression by RT-qPCR shown that the expression of the gip gene is higher when the pathogen grows in the presence of 0.2 % sawdust (w/v) as a inductor substract. The essential oils extracted from Mentha pulegium L. revealed that P. cinnamomi is inhibited in a concentration of 80 % (v/v), in which the plant remains viable and its survival is not afected. By this way the use of this natural product as an active agent if of great importance, specially in the regions that have chestnut orchards as natural resources, with high economical value, and may ultimately be an alternative way in the control of P. cinnamomi.

Mestrado em Engenharia Agronómica - especialização em Engenharia Rural - Instituto Superior de Agronomia
Desde o século passado tem-se vindo a registar um aumento da mortalidade de sobreiros, que tem vindo a ser associado a Phytophthora cinnamomi, que é considerado um fator determinante do declínio do montado. Como os tratamentos testados no controlo desta doença têm demonstrado resultados insatisfatórios, pretendeu-se, analisar se a aplicação de produtos não convencionais poderá alterar as perspetivas de controlo da doença no tratamento de Quercus suber infetados com P. cinnamomi. Com a finalidade de avaliar a agressividade de cinco isolados de P. cinnamomi, efetuaram-se ensaios de inoculação em estacas de sobreiro e em Trifolium subterraneum L. Pretendeu-se selecionar o isolado que induzisse, de uma forma mais rápida, um maior grau de lesões radiculares no mais curto período de tempo. Testaram-se produtos com propriedades bioestimulantes das plantas e/ou antagonistas do oomiceta no controlo de P. cinnamomi em sobreiro: ácido salicílico, que estimula as respostas de defesa da planta; BLAD, uma proteína multifuncional com propriedades antifúngicas e bioestimulantes, e extrato de Omphalotus olearius, que tem um efeito antagonista em relação a P. cinnamomi. O fosfonato de potássio também foi usado como referência, visto já ter demonstrando resultados positivos em Quercus spp.
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During the initiation and execution of a rootstock breeding programme to overcome the financially crippling disease, Phytophthora root rot of avocado, various constraints have been identified for both the breeding as well as the screening aspect of the programme. A review of the literature revealed a complex host-pathogen interaction that should be taken into account in the recombination and screening of genetic material. With the detection of beneficial genotypes being the crux of a breeding programme, this dissertation was focused on the screening of rootstock material for tolerance to Phytophthora cinnamomi. Screening should be scientific but at the same time also be time and cost effective. Specific attention was given to (i) the correct medium for screening mass numbers of seedlings, (ii) fast and effective cloning of single selections, and (iii) evaluation of clonal material for tolerance to P. cinnamomi. Soil as a screening medium was compared with three inert hydroponic media as well as one aeroponic system. Only soil was found to be ineffective due to its properties. The other media tested, namely, sand, vermiculite, water and the aeroponic system were equal in performance. The medium to be used will depend on the preference of the breeder as each medium has its own pro's and con's. It was, however, found that the evaluation criterion to be applied depends on the medium that is used. With regard to cloning of single selections, a definite difference with regard to the cloning ability of the different selections was found. An inability to be etiolated was displayed by some of the selections and these could thus not be vegetatively propagated and were not further tested. One of the tolerance mechanisms in the standard cultivar Duke 7, is root regeneration. It was thus expected that this characteristic cloning would give an indication of the rootstock's ability to tolerate P. cinnamomi. This could not be confirmed, but most of the selections did, however, perform better than Duke 7. Comparison of feeder root percentage in non-inoculated and inoculated treatments was not sufficient for facilitating the final selection of candidate rootstocks from a large number of potential clonal selections. Four selections were made, based on the hypothesis that a larger root system will be a better forager and thus enhance the horticultural aspects of the rootstock-scion combination. Valuable information was obtained with regard to various mediums and criteria to be used during mass screening and final screening of clonal selections. This knowledge must be taken into account in the planning of future breeding projects. During this project a total of 38 984 seedlings were screened and four selections were made. For both the nursery and the producer, knowledge of the clonal ability of a potential new rootstock is important from a financial point of view.
Dissertation (MSc (Horticultural Science))--University of Pretoria, 2006.
Plant Production and Soil Science
unrestricted

This study focuses on the soil- and water-borne plant pathogen Phytophthora cinnamomi Rands and the phenomenon of P. cinnamomi suppressive soil. In particular, this thesis reports on the outcome of field surveys and glasshouse assays undertaken to locate P. cinnamomi suppressive soils and to confirm the involvement of biological processes in suppression. The potential role of cellulase and laminarinase in suppression was investigated and a molecular technique known as length heterogeneity PCR (LH-PCR) was used to analyse the structure and diversity of bacterial and fungal communities in avocado orchard soils that were suppressive and conducive to P. cinnamomi. Four avocado orchards with P. cinnamomi suppressive soils were identified and soils were ã-irradiated to destroy their suppressive capacity, thus confirming biological suppression. Suppression was also partially transferred to ã-irradiated and conducive soils by mixing with 10% suppressive avocado soils. Cellulase and laminarinase activities measured in avocado orchard soils inoculated with P. cinnamomi were not associated with disease severity in lupin seedlings during glasshouse assays involving the same soil samples. Minor shifts in bacterial and fungal community structure were observed in response to mixing conducive and irradiated soils with suppressive soils. This was associated with decreased disease severity in avocado seedlings in these treatments. The shift in bacterial community structure was partially determined by the appearance and increased abundance of several bacterial 16S rDNA sequences, which were unique to the suppressive soils, in the mixed soil treatments. It is suggested that the bacteria and fungi from which these sequences originated may be involved in suppression and further work should be undertaken to determine their identity and confirm their potential role in the development and maintenance of P. cinnamomi suppressive soils.
Doctor of Philosophy (PhD)

This study focuses on the soil- and water-borne plant pathogen Phytophthora cinnamomi Rands and the phenomenon of P. cinnamomi suppressive soil. In particular, this thesis reports on the outcome of field surveys and glasshouse assays undertaken to locate P. cinnamomi suppressive soils and to confirm the involvement of biological processes in suppression. The potential role of cellulase and laminarinase in suppression was investigated and a molecular technique known as length heterogeneity PCR (LH-PCR) was used to analyse the structure and diversity of bacterial and fungal communities in avocado orchard soils that were suppressive and conducive to P. cinnamomi. Four avocado orchards with P. cinnamomi suppressive soils were identified and soils were ã-irradiated to destroy their suppressive capacity, thus confirming biological suppression. Suppression was also partially transferred to ã-irradiated and conducive soils by mixing with 10% suppressive avocado soils. Cellulase and laminarinase activities measured in avocado orchard soils inoculated with P. cinnamomi were not associated with disease severity in lupin seedlings during glasshouse assays involving the same soil samples. Minor shifts in bacterial and fungal community structure were observed in response to mixing conducive and irradiated soils with suppressive soils. This was associated with decreased disease severity in avocado seedlings in these treatments. The shift in bacterial community structure was partially determined by the appearance and increased abundance of several bacterial 16S rDNA sequences, which were unique to the suppressive soils, in the mixed soil treatments. It is suggested that the bacteria and fungi from which these sequences originated may be involved in suppression and further work should be undertaken to determine their identity and confirm their potential role in the development and maintenance of P. cinnamomi suppressive soils.

Dissertação de Mestrado em Genética Molecular Comparativa e Tecnológica
Phytophthora cinnamomi é um fitopatogéneo do solo altamente difundido que está associado à doença da tinta dos castanheiros. Apesar dos esforços para controlo desta doença através da aplicação de fungicidas específicos, estes não se têm revelado eficazes. Assim, os agentes de controlo biológico (BCA) têm-se tornado uma alternativa de crescente interesse aos fungicidas. Este trabalho teve como objectivo o isolamento e screening de isolados bacterianos com actividade antagonista para com P. cinnamomi. A actividade antagonista foi avaliada in vitro tendo sido estudados um total de 339 isolados. Apenas 12% destes isolados demonstraram uma boa actividade antagonista (≥50%) nos testes em cocultura. Todos os isolados foram analisados quanto à produção de enzimas hidrolíticas, sideróforos e solubilização do fosfato. A diversidade genética foi estudada através da análise dos perfis de M13, PCR-RFLP e a análise filogenética através da sequenciação da região 16S rDNA. Os géneros Bacillus, Paenibacillus, Pseudomonas e Streptomyces foram os maioritariamente obtidos nos testes de antagonismos. Os isolados antagonistas foram subsequentemente seleccionados para inoculação em bioensaios de sorgo e milho de modo a verificar a interacção e influência destes microrganismos no crescimento das plantas. Devido aos seus resultados, os isolados BC7, B106, B167A, B20-B, BC8-B, BC133 e BC27 foram considerados potenciais promotores do crescimento das plantas e promissores BCAs para controlo da doença da tinta dos castanheiros.
Phytophothora cinnamomi is one of the most widespread soil-borne pathogen and is associated with the chestnut ink disease. Although efforts are being made to control the disease using specific fungicides, they are not been effective. Biological control agents (BCA) are becoming an increasingly interesting alternative to chemical fungicides. The aims of this study were isolate and screen the bacterial strains showing highest antifungal activity against P. cinnamomi. A total of 339 bacterial strains were isolated and tested for the in vitro antagonism against P.cinnamomi. Among these isolates, only 12 bacterial strains exhibited a maximum antagonistic activity in dual culture assay (≥50%). The isolates where also tested for the production of hydrolytic enzymes, siderophores and phosphorous solubilization. The genetic diversity of the selected isolates was studied by M13 analysis, PCR-RFLP and phylogenetic analysis of 16S rDNA sequences. Based on the 16S rDNA sequencing, Bacillus, Paenibacillus, Pseudomonas and Streptomyces were the most in vitro antagonist isolates identified. Isolates were further assayed in greenhouse bioassays with maize and sorghum plants for investigation of their interaction and influence on plant´s growth. Among these isolates the BC7, B106, B167A, B20-B, BC8-B, BC133 and BC27 are considered potential plant growth promoters and also promissory BCAs for chestnut ink disease.

Tese de doutoramento, Ciências Agrárias (Proteção de Plantas), Faculdade de Ciência e Tecnologia, Universidade do Algarve, 2014
Phytophthora cinnamomi Rands devastates natural ecosystems and crops around the world causing enormous economic losses. The “montado” ecosystem is threatened by this highly aggressive pathogen. Much concern involving fungicide use highlighted the need to develop new environmentally friendly means of control. In this work, Phlomis purpurea L., which was recently reported to have activity against P. cinnamomi and protect susceptible Quercus ilex and Q. suber from infection, was further studied. To determine the protective activity of P. purpurea on the infection of susceptible species a greenhouse assay was implemented showing that, when planted next to the host, it was able to significantly reduce the average severity of host’s root symptoms. A field assay was set on naturally infested plots to determine the protective effect of this herbaceous plant towards Q. suber. P. purpurea significantly increased the emergence and survival of Q. suber. In both assays P. purpurea planted alone was able to completely eliminate P. cinnamomi. In order to study the effectiveness of P. purpurea root extracts (PRE) to control Q. suber and Q. ilex root infection by this oomycete, in vivo assays were set up. PRE at 10 mg ml-1 significantly inhibited P. cinnamomi zoospore infection of both plants. Moreover, PRE was able to elicit a defence response on Q. suber roots. To identify the active principle, bioactivity guided isolation was performed after fractionation of PRE. Isolation, purification and structural characterization led to identification of a novel nortriterpene, phlomispurpentaolone, containing the anti-P. cinnamomi activity. Moreover, P. purpurea metabolites produced constitutively and upon challenge with P. cinnamomi were quantified using LC-MS showing that phlomispurpentaolone is a phytoanticipin. Another goal was to confirm the resistance of P. purpurea to P. cinnamomi by histological techniques. P. purpurea roots were shown to have a constitutive strengthened exodermis that can act, per se, as a physical barrier for the penetration of P. cinnamomi.
Phytophthora cinnamomi Rands é um oomiceta que, tendo uma vasta gama de hospedeiros devasta ecossistemas naturais e culturas em todo mundo, causando enormes prejuízos económicos. O "montado" é um ecossistema agro-silvo-pastoril de alto valor socioeconómico, composto principalmente por sobreiros (Quercus suber) e azinheiras (Quercus ilex subsp. rotundifolia) que estão a ser dizimados por este agente patogénico. O sobreiro reveste-se de particular importância visto que é a árvore de onde se extrai a cortiça. Portugal é o primeiro produtor e exportador de cortiça mas essa posição está em risco dado que os sobreiros estão a ser substituídos por outras árvores mais resistentes a P. cinnamomi. Sendo um agente patogénico do solo dotado de grande capacidade de sobrevivência P. cinnamomi é extremamente difícil de erradicar, incluíndo o uso dos fungicidas. A grande preocupação, quer ambiental quer para a saúde, que envolve o uso de fungicidas levou à necessidade de se desenvolverem novos meios ecológicos de controlo. A marioila (Phlomis purpurea L.) é uma herbácea, da família Lamiaceae, espontânea em habitats de Quercus suber e Q. ilex subsp. rotundifolia no sul de Portugal que cresce, sem manifestar sinais de doença, em áreas afetadas pela doença declínio. Recentemente, foi por nós publicado que o extrato de raízes de Phlomis purpurea (PRE) inibe significativamente a produção de todas as estruturas de ciclo da doença bem como a germinação de clamidósporos e de zoósporos. Também foi demonstrado em ensaios de estufa que P. purpurea reduz o potencial do inóculo no solo, o que se traduz na capacidade de reduzir a infeção de outras plantas, sugerindo que P. purpurea tem potencial para evitar a disseminação da doença. Esta planta é resistente à infeção por este agente patogénico. Neste trabalho, foram realizados ensaios de estufa para determinar a atividade protetora de P. purpurea na infeção de Quercus canariensis, Q. coccifera e Castanea sativa quando é co-plantada com cada uma delas num mesmo vaso. P. purpurea reduziu significativamente a severidade média dos sintomas radiculares das plantas hospedeiras. Foi, também, efetuado um ensaio de campo em parcelas naturalmente infestadas com P. cinnamomi para determinar o efeito protetor desta planta em Q. suber. Em duas parcelas foram semeadas landes de sobreiro e em outras duas parcelas landes de sobreiro juntamente com P. purpurea. Esta planta aumentou significativamente a germinação e consequentemente a emergência e a sobrevivência de Q. suber. Em ambos os ensaios P. purpurea isolada foi, por si só, capaz de eliminar completamente o inóculo de P. cinnamomi presente no solo à volta das raízes. De modo a estudar a eficácia do PRE no controlo da infeção das raízes de Q. suber e Q. ilex por parte deste oomiceta, foram realizados ensaios in vivo. O PRE, à concentração de 10 mg ml-1, inibiu significativamente (84.1%) a infeção pelos zoósporos de P. cinnamomi em ambas as plantas, não sendo as raízes de Q. ilex sequer infetadas. Além disso, o PRE foi capaz de provocar uma resposta de defesa nas raízes de Q. suber, como revelado pela redução significativa da proporção de raízes infetadas. Outro objetivo foi estudar a interacção de P. purpurea com P. cinnamomi utilizando técnicas histológicas. As raízes de P. purpurea infestadas com zoósporos e micélio, após 72 h, não apresentaram sinais de infeção. Também não apresentaram alterações histológicas. Algumas poucas hifas foram detetadas na superfície da epiderme mas não conseguiram penetrar na raiz. As raízes de marioila mostraram ter uma epiderme reforçada, com lignina e taninos, e uma exoderme, composta por celulose e suberina, que pode atuar, por si, como uma barreira física impedindo a penetração de P. cinnamomi. Por outro lado, infestou-se Q. suber (uma espécie susceptível) com micélio e zoósporos. Após 16-24 h as raízes começaram a mostrar sinais de infeção e após 72 h apresentavam necroses extensas. Após 48 h eram visíveis, microscopicamente, danos nos tecidos e distorção da parede celular com proliferação das hifas tanto inter como intra-celularmente, através da epiderme, do parênquima cortical e cilindro vascular. Porém, as raízes eliciadas com PRE, 24 h antes da infestação com o agente patogénico, apresentavam células epidérmicas e hipodérmicas irregulares com paredes espessas pela deposição de lignina e taninos e, 48 h após a infestação com micélio e zoósporos, as hifas apresentavam-se restritas principalmente aos espaços intercelulares da epiderme, com pequenas incursões no córtex mas não alcançaram o cilindro vascular. Além disso, as raízes de Q. suber expostas simultaneamente a PRE e zoósporos, não mostraram lesões macroscópicas após 48 horas e, também não foi observada penetração nos tecidos ao microscópio. Provavelmente, esta proteção foi causada pela atividade antagónica direta do PRE sobre os zoósporos como mostrado previamente in vitro. Um dos objetivos deste estudo foi, ainda, identificar uma substância, eventualmente, responsável pela inibição de P. cinnamomi. O PRE foi analisado por HPLC-ESI/MS/MS. As frações de HPLC foram recuperadas e a sua actividade anti-P. cinnamomi determinada. A fração com maior atividade revelou a presença de um composto principal com m/z 490,4 (M+H)+, que inibiu completamente o crescimento de micélio de P. cinnamomi a uma concentração de 0,5 mg ml-1. Este composto foi isolado a partir de PRE por cromatografia preparativa em sílica gel com uma pureza de ca 90 % e foi purificado por recristalização Este é um novo nortriterpenoide estruturalmente caracterizado por ressonância magnética nuclear (RMN) 1D e 2D, espectrometria de massa por ionização por electropulverização (ESI/MS), espectroscopia de infravermelho (IR) e difração de raios-X (XRD). Este composto foi denominado phlomispurpentaolone. A uma concentração de 0,1 mgml-1, phlomispurpentaolone inibiu 75,7 % do crescimento do micélio de P. cinnamomi, cerca de 100 vezes mais do que o PRE. Com a finalidade de descobrir outros metabolitos que são potencialmente bioativos contra P. cinnamomi, plântulas de P. purpurea foram inoculadas com zoósporos em 6 tempos (0, 6, 12, 24, 48 e 72 h). Foram, também, preparados controlos e exsudatos das mesmas plantas nos mesmos intervalos de tempo. O material a partir das plantas em cada intervalo de tempo foi reunido (11 x 5 repetições). As raízes e as folhas foram extraídas com MeOH. Lípidos e metabolitos ligeiramente polares foram separados utilizando cromatografia de fase reversa (RPC) com HSS T3 C18. Os exsudados também foram recolhidos e esterilizados por filtração a cada intervalo de tempo, imediatamente submersos em azoto líquido e mantidos a -80 °C. Os metabolitos de P. purpurea produzidos, tanto constitutivamente como após infestação com P. cinnamomi, foram quantificados utilizando um sistema de cromatografia líquida acoplada a espectrometria de massa (UPLC-MS). Os exsudatos das raízes foram analisados por cromatografia gasosa acoplada a espectrometria de massa (GC-MS) após derivatização. Verificou-se que phlomispurpentaolone é produzido constitutivamente. Este trabalho de investigação contribuiu para elucidar os mecanismos de defesa de P. purpurea contra P. cinnamomi bem como o principal composto produzido por esta planta com atividade contra este agente patogénico. A capacidade de erradicar este agente patogénico utilizando uma planta autóctone abre novas perspectivas para o controlo do declínio do sobreiro bem como de outras espécies hospedeiras deste oomiceta.

Tese dout., Ciências Agrárias, Universidade do Algarve, 2007
Estudou-se a evolução do declínio de sobreiros (Quercus suber) e azinheiras (Q. rotundifolia) em Portugal e na província de Andaluzia em Espanha. A observação do sistema radicular das árvores afectadas, revelou muitas raízes necróticas e elevadas percentagens de raízes absorventes mortas. Das raízes, assim como do solo associado, isolou-se o oomicete Phytophthora cinnamomi, patogénio radicular de numerosas espécies lenhosas, entre as quais se encontram espécies mediterrâneas do género Quercus. Registou-se, em diferentes condições edafo-climáticas, a progressão da incidência e da severidade do declínio de Q. suber, que aumentaram progressivamente, ao longo de 5 anos. O estudo de crescimento in vitro dos isolamentos de P. cinnamomi obtidos nas prospecções em sobreirais e montados, permitiu determinar temperaturas óptimas de crescimento bastante elevadas (26.9 – 30,1ºC). O desenvolvimento da infecção por P. cinnamomi foi mais severo em condições de permanente saturação hídrica do solo. A temperatura não influenciou a infecção. O método semi-quantitativo da avaliação visual de sintomas foi o que melhor expressou a severidade da doença, pois permitiu examinar a funcionalidade das raízes, que não era possível ser detectada com os métodos quantitativos ensaiados. Avaliou-se o efeito de diferentes substâncias químicas no controlo da infecção em sobreiros e azinheiras jovens e sobreiros adultos, tendo-se verificado uma grande variabilidade de eficácia entre si.
Fundação para a Ciência e a Tecnologia (FCT)