Cytological effects of cellulases in the parasitism of Phytophthora parasitica by Pythium oligandrum

The ubiquitous oomycete Pythium oligandrum is a potential biocontrol agent for use against a wide range of pathogenic fungi and an inducer of plant disease resistance. The ability of P. oligandrum to compete with root pathogens for saprophytic colonization of substrates may be critical for pathogen increase in soil, but other mechanisms, including antibiosis and enzyme production, also may play a role in the antagonistic process. We used transmission electron microscopy and gold cytochemistry to analyze the intercellular interaction between P. oligandrum and Phytophthora parasitica. Growth of P. oligandrum towards Phytophthora cells correlated with changes in the host, including retraction of the plasma membrane and cytoplasmic disorganization. These changes were associated with the deposition onto the inner host cell surface of a cellulose-enriched material. P. oligandrum hyphae could penetrate the thickened host cell wall and the cellulose-enriched material, suggesting that large amounts of cellulolytic enzymes were produced. Labeling of cellulose with gold-complexed exoglucanase showed that the integrity of the cellulose was greatly affected both along the channel of fungal penetration and also at a distance from it. We measured cellulolytic activity of P. oligandrum in substrate-free liquid medium. The enzymes present were almost as effective as those from Trichoderma viride in degrading both carboxymethyl cellulose and Phytophthora wall-bound cellulose. P. oligandrum and its cellulolytic enzymes may be useful for biological control of oomycete pathogens, including Phytophthora and Pythium spp., which are frequently encountered in field and greenhouse production.     

Trichoderma harzianum might impact phosphorus transport by arbuscular mycorrhizal fungi

Trichoderma sp. is a biocontrol agent active against plant pathogens via mechanisms such as mycoparasitism. Recently, it was demonstrated that Trichoderma harzianum was able to parasitize the mycelium of an arbuscular mycorrhizal (AM) fungus, thus affecting its viability. Here, we question whether this mycoparasitism may reduce the capacity of Glomus sp. to transport phosphorus ((33)P) to its host plant in an in vitro culture system. (33)P was measured in the plant and in the fungal mycelium in the presence/absence of T. harzianum. The viability and metabolic activity of the extraradical mycelium was measured via succinate dehydrogenase and alkaline phosphatase staining. Our study demonstrated an increased uptake of (33)P by the AM fungus in the presence of T. harzianum, possibly related to a stress reaction caused by mycoparasitism. In addition, the disruption of AM extraradical hyphae in the presence of T. harzianum affected the (33)P translocation within the AM fungal mycelium and consequently the transfer of (33)P to the host plant. The effects of T. harzianum on Glomus sp. may thus impact the growth and function of AM fungi and also indirectly plant performance by influencing the source-sink relationship between the two partners of the symbiosis.     

Interaction of the mycoparasite Pythium oligandrum with other Pythium species

Interactions of Pythium oligandrum and four plant‐pathogenic Pythium spp. (P. ultimum, P. vexans, P. graminicola and P. aphanidermatum,) were studied in vitro by (i) video microscopy of hyphal interactions on water agar films, (ii) counting of host and mycoparasite propagules in different regions of opposing colonies on sunflower‐seed extract agar films and (Hi) ability of P. oligandrum to overgrow plates of potato‐dextrose agar previously colonized by Pythium spp. Pythium oligandrum typically coiled round the hyphae of Pythium hosts and penetrated the host hyphae after approximately 50 min from the hyphal coils, causing disruption of host hyphal tips up to 1.2 mm ahead of contact points. The relative growth rates of mycoparasite and host hyphae, timing of penetration and distance (sub‐apical) at which penetration led to host tip disruption were used to assess the potential of mycoparasitism by P. oligandrum to prevent the growth of Pythium hosts. P. aphanidermatum was unique among the ‘host’ Pythium spp. in being largely unaffected by P. oligandrum and in antagonizing the mycoparasite by coiling and penetrating the mycoparasite hyphae. Other host Pythium spp. apparently differed in susceptibility, the most susceptible being P. vexans and P. ultimum, whereas P. graminicola was more resistant. The results are discussed in relation to the role of P. oligandrum as a biocontrol agent, especially for limiting the ability of other Pythium spp. to increase their propagule populations in crop residues.     

Rhizosphere persistence of three Pythium oligandrum strains in tomato soilless culture assessed by DNA macroarray and real-time PCR

In tomato soilless culture, plant-disease optimal control and growth promotion are achieved when the rhizosphere is heavily colonized by the biocontrol agent Pythium oligandrum. Discrepancies in performance are generally attributed to the poor persistence of P. oligandrum on roots. In this study, three selected strains of P. oligandrum were introduced into the rhizosphere of greenhouse-grown tomato plants, and their persistence was assessed by DNA macroarray hybridization and real-time PCR. The experimental data from DNA detection and plate counting were compared. PCR-based methods detected P. oligandrum throughout the 6-month growing season, whereas plate counting indicated its presence only over the first 3 months. Moreover, the DNA array method provided information about the various Pythium species present in the rhizosphere: P. dissotocum was frequently detected on roots of plants, without distinction between plants inoculated or not inoculated with the antagonist. The detection of other Pythium species was noticed sporadically (P. ultimum, P. sylvaticum and P. intermedium), independent of the treatment. Even though the yield enhancement is not significant throughout the entire growing season, data obtained from epidemiological studies demonstrate an enhancement of P. oligandrum persistence on the rhizosphere of plants and less use of mycoparasitism.     

Characterisation of the early events in atypical tomato root colonisation by a biocontrol agent, Pythium oligandrum.

The specific oomycete-plant relationship established between a biological agent, Pythium oligandrum, and tomato (Lycopersicon esculentum Mill.) plants was examined over the first 48 h after inoculation of tomato roots with the antagonist. One of the most significant effects was the quick colonisation of cortical and vascular root areas by P. oligandrum (until 9 h post-inoculation); it was similar to invasions by the major pathogens of Pythium genus, and much faster than those by Pythium-minor pathogens. Despite the multiplication of hyphae in the root areas, fungal colonisation was associated with neither host wall disruption nor host cell alterations. The colonising hyphae looked healthy till the ninth hour after inoculation, then, they progressively became highly vacuolated. Cytological observations showed that, over the first 14 h of experiment, oomycete invasion was accompanied with rare host-induced defence reactions. Biochemical analysis evidenced an accumulation of phenolic compounds starting 3 h after inoculation. The 14th hour corresponded to the beginning of rishitin (phytoalexin) synthesis. Accumulation of biochemical host defence compounds was concomitant with early signs of hyphae alterations. During the next 34 h several host reactions were regularly amplified as evidenced by the plugging of invaded host cells with heterogeneous osmiophilic or high electron-dense (ED) materials. Fungal cell decay was accompanied with the formation of oogonia in the cortex, vascular parenchyma and xylem vessels. All these early events suggest a peculiar relationship established between P. oligandrum and the plant.     

A novel Phytophthora infestans haustorium-specific membrane protein is required for infection of potato

Phytophthora infestans causes late-blight, a devastating and re-emerging disease of potato crops. During the early stages of infection, P. infestans differentiates infection-specific structures such as appressoria for host epidermal cell penetration, followed by infection vesicles, and haustoria to establish a biotrophic phase of interaction. Here we report the cloning, from a suppression subtractive hybridization library, of a P. infestans gene called Pihmp1 encoding a putative glycosylated protein with four closely spaced trans-membrane helices. Pihmp1 expression is upregulated in germinating cysts and in germinating cysts with appressoria, and significantly upregulated throughout infection of potato. Transient gene silencing of Pihmp1 led to loss of pathogenicity and indicated involvement of this gene in the penetration and early infection processes of P. infestans. P. infestans transformants expressing a Pihmp1::monomeric red fluorescent protein (mRFP) fusion demonstrated that Pihmp1 was translated in germinating sporangia, germinating cysts and appressoria, accumulated in the appressorium, and was located at the haustorial membrane during infection. Furthermore, we discovered that haustorial structures are formed over a 3 h period, maturing for up to 12 h, and that their formation is initiated only at sites on the surface of intercellular hyphae where Pihmp1::mRFP is localized. We propose that Pihmp1 is an integral membrane protein that provides physical stability to the plasma membrane of P. infestans infection structures. We have provided the first evidence that the surface of oomycete haustoria possess proteins specific to these biotrophic structures, and that formation of biotrophic structures (infection vesicles and haustoria) is essential to successful host colonization by P. infestans.     

The malarial host-targeting signal is conserved in the Irish potato famine pathogen

Animal and plant eukaryotic pathogens, such as the human malaria parasite Plasmodium falciparum and the potato late blight agent Phytophthora infestans, are widely divergent eukaryotic microbes. Yet they both produce secretory virulence and pathogenic proteins that alter host cell functions. In P. falciparum, export of parasite proteins to the host erythrocyte is mediated by leader sequences shown to contain a host-targeting (HT) motif centered on an RxLx (E, D, or Q) core: this motif appears to signify a major pathogenic export pathway with hundreds of putative effectors. Here we show that a secretory protein of P. infestans, which is perceived by plant disease resistance proteins and induces hypersensitive plant cell death, contains a leader sequence that is equivalent to the Plasmodium HT-leader in its ability to export fusion of green fluorescent protein (GFP) from the P. falciparum parasite to the host erythrocyte. This export is dependent on an RxLR sequence conserved in P. infestans leaders, as well as in leaders of all ten secretory oomycete proteins shown to function inside plant cells. The RxLR motif is also detected in hundreds of secretory proteins of P. infestans, Phytophthora sojae, and Phytophthora ramorum and has high value in predicting host-targeted leaders. A consensus motif further reveals E/D residues enriched within approximately 25 amino acids downstream of the RxLR, which are also needed for export. Together the data suggest that in these plant pathogenic oomycetes, a consensus HT motif may reside in an extended sequence of approximately 25-30 amino acids, rather than in a short linear sequence. Evidence is presented that although the consensus is much shorter in P. falciparum, information sufficient for vacuolar export is contained in a region of approximately 30 amino acids, which includes sequences flanking the HT core. Finally, positional conservation between Phytophthora RxLR and P. falciparum RxLx (E, D, Q) is consistent with the idea that the context of their presentation is constrained. These studies provide the first evidence to our knowledge that eukaryotic microbes share equivalent pathogenic HT signals and thus conserved mechanisms to access host cells across plant and animal kingdoms that may present unique targets for prophylaxis across divergent pathogens.     

Pseudozyma aphidis activates reactive oxygen species production,programmed cell death and morphological alterations in the necrotrophic fungus Botrytis cinerea

Many types of yeast have been studied in the last few years as potential biocontrol agents against different phytopathogenic fungi. Their ability to control plant diseases is mainly through combined modes of action. Among them, antibiosis, competition for nutrients and niches, induction of systemic resistance in plants and mycoparasitism have been the most studied. In previous work, we have established that the epiphytic yeast Pseudozyma aphidis inhibits Botrytis cinerea through induced resistance and antibiosis. Here, we demonstrate that P. aphidis adheres to B. cinerea hyphae and competes with them for nutrients. We further show that the secreted antifungal compounds activate the production of reactive oxygen species and programmed cell death in B. cinerea mycelium. Finally, P. aphidis and its secreted compounds negatively affect B. cinerea hyphae, leading to morphological alterations, including hyphal curliness, vacuolization and branching, which presumably affects the colonization ability and infectivity of B. cinerea. This study demonstrates additional modes of action for P. aphidis and its antifungal compounds against the plant pathogen B. cinerea.     

Comparison of real-time PCR and microscopy to evaluate sclerotial colonisation by a biocontrol fungus

The biocontrol agent Trichoderma harzianum colonises sclerotia of the plant pathogenic fungus Sclerotinia sclerotiorum. Plating of sclerotia typically has been used to determine the incidence of mycoparasitism, but does not quantify the extent to which individual sclerotia are colonised. We developed a specific PCR primer/probe set for the green fluorescent protein (GFP)-transformant T. harzianum ThzID1-M3, which exhibited high precision and reproducibility. Quantitative real-time PCR was evaluated along with epifluorescence microscopy and image analysis to investigate dynamics of colonisation of sclerotia in non-sterile soil. Amounts of ThzID1-M3 DNA and S. sclerotiorum DNA from entire individual sclerotia were quantified using real-time PCR. Epifluorescence micrographs were captured from sclerotial thin-section samples, and GFP fluorescence from these was quantified using computer image analysis in order to estimate colonisation on a per-sclerotium basis. As determined by either method, ThzID1-M3 colonised sclerotia in soil, and both methods quantified colonisation dynamics over time. In a separate experiment, colonisation of sclerotia on agar plates was observed using confocal laser scanning microscopy to view the GFP-fluorescing hyphae of ThzID1-M3. This method, while highly labour-intensive, provided high spatial resolution of colonisation dynamics. Thus, each method has advantages: microscopy combined with image analysis can provide useful information on the spatial and temporal dynamics of colonisation, while real-time PCR can provide a more precise assessment of the extent of sclerotial colonisation over time and can more easily be used to sample entire sclerotia.     

Ecofriendly control of potato late blight causative agent and the potential role of lactic acid bacteria: a review

In times of increasing societal pressure to reduce the application of pesticides on crops, demands for environmentally friendly replacements have intensified. In the case of late blight, a devastating potato plant disease, the historically most widely known plant destroyer has been the oomycete Phytophthora infestans. To date, the most important strategy for control of this pathogen has been the frequent application of fungicides. Due to the aforementioned necessity to move away from traditional chemical treatments, many studies have focused on finding alternative ecofriendly biocontrol systems. In general, due to the different modes of actions (i.e. antagonistic effects or induction of plant defence mechanisms), the use of microorganisms as biological control agents has a definite potential. Amongst them, several species of lactic acid bacteria have been recognised as producers of bioactive metabolites which are functional against a broad spectrum of undesirable microorganisms, such as fungi, oomycetes and other bacteria. Thus, they may represent an interesting tool for the development of novel concepts in pest management. This review describes the present situation of late blight disease and summarises current literature regarding the biocontrol of the phytopathogen P. infestans using antagonistic microorganisms.     

Physical mapping across an avirulence locus of Phytophthora infestans using a highly representative, large-insert bacterial artificial chromosome library

The oomycete plant pathogen Phytophthora infestans is the causal agent of late blight, one of the most devastating diseases of potato worldwide. As part of efforts to clone avirulence (Avr) genes and pathogenicity factors from P. infestans, we have constructed a bacterial artificial chromosome (BAC) library from an isolate containing six Avr genes. The BAC library comprises clones with an average insert size of 98 kb and represents an estimated 10 genome equivalents. A three-dimensional pooling strategy was developed to screen the BAC library for amplified fragment length polymorphism (AFLP) markers, as this type of marker has been extensively used in construction of a P. infestans genetic map. Multiple positive clones were identified for each AFLP marker tested. The pools were used to construct a contig of 11 BAC clones in a region of the P. infestans genome containing a cluster of three avirulence genes. The BAC contig is predicted to encompass the Avr11 locus but mapping of the BAC ends will be required to determine if the Avr3 and Avr10 loci are also present in the BAC contig. These results are an important step towards the positional cloning of avirulence genes from P. infestans, and the BAC library represents a valuable resource for largescale studies of oomycete genome organisation and gene content.     

Effect of proteinaceous proteinase inhibitors from potato tubers on the growth and development of phytopathogenic microorganisms

We studied the effect of two proteins, PSPI-21 and PKSI, on the growth and development of phytopathogenic microorganisms (Phytophthora infestans oomycete and Fusarium culmorum fungus). Both proteins were isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii) and served as inhibitors of serine proteinases. These proteins differed in the ability to inhibit growth of Phytophthora infestans oomycete and Fusarium culmorum fungus. PSPI-21 was the most potent in modulating the growth of oomycete mycelium. PKSI primarily affected the growth of the fungal mycelium. The proteins under study induced complete destruction of oomycete zoospores and partial destruction of fungal macroconidia. Our results suggest that these proteins are involved in the protection of potato plants from phytopathogenic microorganisms.     

基于PEG介导原生质体转化构建粉红聚端孢荧光标记

粉红聚端孢是多种植物的重要病原菌。本研究通过酶解粉红聚端孢幼嫩菌丝细胞壁获得原生质体,用PEG介导原生质体转化将携带GFP基因和博来霉素抗性基因的外源DNA随机插入粉红聚端孢基因组,共获得博来霉素抗性菌株90株,转化效率达18个/μg。选取22株转化子荧光观察,发现均可表达荧光,菌株间荧光强度不同,其中10株转化子荧光表达较强。与野生型相比,突变菌株TR45的菌落生长、产孢量和致病力等生物学特性均未改变,在不含博来霉素的培养皿中继代培养10代荧光仍能稳定表达。本研究构建的高效原生质体制备和PEG介导粉红聚端孢遗传转化方法,可用于该菌基因功能研究,绿色荧光标记菌株可用于病菌侵入、田间监测、侵染循环等发生规律研究。     

Pythium periplocum, an aggressive mycoparasite of Botrytis cinerea causing the gray mould disease of grape-vine

Pythium periplocum Dreschler has been found to be an aggressive mycoparasite of Botrytis cinerea, the causal agent of the gray mould disease of the grape-vine. When grown together, the former enters the latter's mycelium, branches freely within, coagulates its cytoplasm and finally tears its hyphae apart, bringing about widespread destruction of the grape-vine pathogen. Extensive coiling around the host, as reported in the case of other mycoparasites belonging to the genus Pythium, has not been observed here. The infected mycelium of B. cinerea fails to infect the grape-vine and does not induce the characteristic gray mould symptoms. Since P. periplocum is not a grape-vine parasite, it could be useful for the biological control of B. cinerea. A brief account of this mycoparasitism is discussed in this article.     

GFP technology for the study of biocontrol agents in tritrophic interactions: a case study with Pseudozyma flocculosa

GFP technology was applied to the biocontrol agent (BCA) Pseudozyma flocculosa to study its development and interactions at the tritrophic level plant-powdery mildew-BCA. Transformation experiments with GFP led to the production of a strongly fluorescent strain, Act-4, that displayed biocontrol traits typical of P. flocculosa WT. Following inundative applications, growth of P. flocculosa Act-4 was closely and almost exclusively associated with the colonies of the pathogen regardless of the powdery mildew species or the host plant tested. Development of P. flocculosa Act-4 on control leaves alone was extremely limited 24 h after its application and was typical of the epiphytic growth characterizing this type of yeast-like fungus. Based on the strong correlation between the colonization pattern of the different powdery mildew species tested and the presence of P. flocculosa Act-4, as determined by its fluorescence, it seems that growth of the BCA is dependant on the presence of powdery mildews. These results demonstrate that the GFP technology can be used to study plant-pathogen-BCA interactions and fulfill a wide array of purposes ranging from fundamental observations of the biocontrol behavior of a BCA to very applied ones serving some of the requirements for the registration of BCA's such as defining their environmental fate.     

Hyphal differentiation in the exploring mycelium of Aspergillus niger

Mycelial fungi play a central role in element cycling in nature by degrading dead organic material such as wood. Fungal colonization of a substrate starts with the invasion of exploring hyphae. These hyphae secrete enzymes that convert the organic material into small molecules that can be taken up by the fungus to serve as nutrients. Using green fluorescent protein (GFP) as a reporter, we show for the first time that exploring hyphae of Aspergillus niger differentiate with respect to enzyme secretion; some strongly express the glucoamylase gene glaA, while others hardly express it at all. When a cytoplasmic GFP was used, 27% of the exploring hyphae of a 5-day-old colony belonged to the low expressing hyphae. By fusing GFP to glucoamylase and by introducing an ER retention signal, this number increased to 50%. This difference is due to cytoplasmic streaming of the reporter in the former case, as was shown by using a photo-activatable GFP. Our findings indicate that a fungal mycelium is highly differentiated, especially when taking into account that hyphae in the exploration zone were exposed to the same nutritional conditions.     

Combining the oomycete Pythium oligandrum with two other antagonistic fungi: Root relationships and tomato grey mold biocontrol

To reduce Pythium oligandrum biocontrol variability and improve its efficacy, experiments were performed by combining the oomycete with two other antagonistic fungi, Fusarium oxysporum strain Fo47 and Trichoderma harzianum. In Petri dishes, Fo47 or T. harzianum hyphae destroyed P. oligandrum cells by antibiosis and mycoparasitism processes; in the rhizosphere of tomato plants (Lycopersicon esculentum), the same antagonistic features were observed. However, in the rhizosphere, hyphae are frequently separated by a certain distance; this allows the coexistence and the persistence of the three microorganisms on the root systems. When introduced in the rhizosphere, Fo47 and P. oligandrum were able to penetrate the root tissues with Fo47 limited to the epidermal and upper layers of cortical cells while P. oligandrum colonized deeper tissue at a faster rate. The two antagonists were killed in few days within roots following elicited plant-defense reactions. T. harzianum was not able to penetrate root tissues. Root colonization with either P. oligandrum alone or in combination with Fo47 and/or T. harzianum resulted in systemic plant resistance which provided plant protection against Botrytis cinerea infection of leaves. The level of control and the expression of pathogenesis-related proteins (PR-proteins) in leaves were similar whatever the antagonistic microbial treatment applied to roots.