Microassay for biological and chemical control of infection of tobacco byPhytophthora parasitica var.nicotianae

We developed a simple, rapid, small-scale assay for infection of tobacco seedlings byPhytophthora parasitica var.nicotianae. One 7-day-old tobacco seedling was placed in each well of a 96-well microtiter plate and inoculated with 500 zoospores ofP. parasitica var.nicotianae. After 72 h all of the inoculated seedlings of the susceptible cultivar, KY14, were infected, and the pathogen had produced sporangia that were visible on the surfaces of the seedlings. Sporangia did not develop on seedlings that were inoculated simultaneously with zoospores and either 1 µg/mL of the chemical fungicide metalaxyl or 5 µL of filtrate of a sporulated culture of the biocontrol agent,Bacillus cereus UW85. Seedlings of tobacco cultivar KY17 were infected byP. parasitica var.nicotianae, although mature plants of this variety are resistant to the pathogen. This microassay may facilitate the rapid screening of potential biological and chemical control agents and may be useful for studying mechanisms of infection and control ofPhytophthora spp. under hydroponic conditions.     

Influence of Fusarium solani on citrus root rot caused by Phytophthora parasitica and Phytophthora citrophthora

Interactions between Fusarium solani and Phytophthora parasitica or F. solani and P. citrophthora influenced the development of root rot of citrus but depended on the temporal order of inoculation with F. solani or the two Phytophthora spp. Inoculation of citrus with either Fusarium solani and Phytophthora parasitica or Phytophthora citrophthora increased root rot compared to inoculation with P. parasitica or P. citrophthora alone when plants were inoculated with Phytophthora by dipping their roots in zoospore suspensions and subsequently transplanted into soil infested with F. solani. However, root rot was not increased by simultaneous co-inoculation of P. parasitica and F. solani or when plants were inoculated with F. solani first. Root rot was not increased when heat-stressed or non-stressed plants were inoculated with P. parasitica 30 days after transplanting into soil infested with F. solani. In most but not all experiments, F. solani alone reduced growth of tops or roots a small but significant amount.Co-inoculation of citrus by root-dipping into zoospore suspensions of P. parasitica and transplanting into soil infested with F. solani reduced feeder root length by 62% and root weight by 61% but did not significantly reduce the percentage of living roots when compared to inoculation with P. parasitica alone. When citrus roots were immersed in zoospore suspensions of P. citrophthora and transplanted into soil infested with F. solani, feeder root length was reduced by 68%, but feeder root weight and the percentage of living roots were not significantly reduced when compared to plants inoculated with P. citrophthora alone.Propagule densities of both P. parasitica and P. citrophthora in the rhizosphere of plants inoculated by root-immersion and then transplanting into soil infested with F. solani were not significantly different than propagule densities from plants transplanted into non-infested soil. Propagule densities of P. parasitica were suppressed an average of 41% when citrus was inoculated with P. parasitica 30 days after transplanting into soil infested with F. solani and by 41% when citrus was co-inoculated by transplanting into soil infested with both F. solani and P. parasitica.     

Distribution of Phytophthora citrophthora R.E. Sm. and E.H. Sm. and P. parasitica Dastur within citrus orchards in Kerman province,Iran

In this research, distribution of Phytophthora species were determined in Kerman Province (Bam, Shahdad and Arzuiyeh) during 2004–2007. The Phytophthora species were isolated from infected root, crown and soil. Root and crown pieces were washed and cultured on a CMA-PARPH medium. The isolation from infected soil was performed by bating method using citrus leaves. It was identified by morphological and several physiological characteristics. Distribution of species was determined by recording the number of isolates recovered from samples from each city. In this study, from 220 soil samples collected from 52 citrus orchards, 80 isolates of Phytophthora were recovered. Among of all isolates of Phytophthora, P. parasitica and P. citrophthora were the most important species of causal agent of gummosis on Citrus sp. Distribution of P. citrophthora was highest in Arzuiyeh and lowest in Bam and Shahdad cities, while distribution of P. parasitica was highest in Bam and Shahdad cities. The majority of soil samples contained only P. parasitica, but a few of percentage samples containing P. citrophthora alone and both of fungi in cites samples.     

In vitro and in vivo antimicrobial efficacy of essential oils and individual compounds against Phytophthora parasitica var. nicotianae

Aim

To evaluate the antimicrobial effects of essential oils (EOs) from cassia, basil, geranium, lemongrass, cumin and thyme, as well as their major components, against Phytophthora parasitica var. nicotianae; to investigate morphological changes in hyphae and sporangia in response to treatment with cinnamaldehyde; and to further evaluate potential biocontrol capacities against tobacco black shank under greenhouse conditions.

Methods and Results

The results revealed that the extent of mycelial growth inhibition was primarily dependent on the composition and concentration of the EOs and the structure of individual compounds. Cinnamaldehyde had a significantly higher inhibitory effect on mycelial growth, formation of sporangia, and production and germination of zoospores in P. parasitica var. nicotianae in vitro, achieving complete inhibition of these phenotypes at 72, 36, 36 and 18 mg l^?1, respectively. Scanning electron microscopic observations revealed that cinnamaldehyde can cause considerable morphological degenerations of hyphae and sporangia such as cytoplasmic coagulation, shrivelled mycelia and sporangia aggregates and swelling and lysis of mycelia and sporangia walls. In vivo assays with cinnamaldehyde demonstrated that this compound afforded protective effect against tobacco black shank under greenhouse conditions in susceptible tobacco plants.

Conclusions

The results of in vitro and in vivo bioassays, together with SEM imaging of the microstructure of P. parasitica var. nicotianae supported the possibility of using cinnamaldehyde as a potent natural biofungicide in the greenhouse.

Significance and Impact of the Study

This study provides a theoretical basis for the potential use of cinnamaldehyde as commercial agents or lead compounds that can be exploited as commercial biofungicides in the protection of tobacco plants from P. parasitica var. nicotianae infection.     

Starch utilization by Phytophthora spp

Summary Phytophthora spp. were grown on artificial starch agar medium. In some cases, the capacity of starch utilization could be a useful tool in species separation ofPhytophthora. Based on the ability to hydrolyse starch,P. palmivora andP. parasitica could be readily distinguished, whereasP. parasitica andP. parasitica var.nicotianae, P. megasperma andP. megasperma var.sojae (P. sojae) behaved similarly. Starch hydrolysis was indicated by a clear unstained zone within the fungal colony when treated with iodine solution. Simple quantitative analysis of starch hydrolysis was made feasible by the following formula:Starch Hydrolysis Index (S.H.I.) = Mean diameter of clear starch hydrolysis zone (d) / Mean diameter of fungal colony (D)     

Phytophthora root-rot of container-grown citrus as affected by foliar sprays and soil drenches of phosphorous and acetyl salicylic acids

Mycelial growth ofPhytophthora nicotianae var.parasitica in vitro was inhibited by phosphorous and acetyl salicylic acids at 10 ppm; mycelial growth ofP. citrophthora was inhibited by phosphorous acid at 10 ppm and acetyl salicylic acid at 100 ppm. Foliar sprays and soil drenches with these chemicals were compared with soil applications of metalaxyl as treatments for Phytophthora root-rot of container-grown citrus (var. Cleopatra mandarin) caused by the aforementioned spp. Foliar sprays with 0.1% phosphorous acid were as effective as the metalaxyl treatment in promoting root growth and phosphorous acid offers considerable potential as an alternative treatment for Phytophthora root-rot of citrus.     

Evaluation of fatty acid methyl ester profile and amplified fragment length polymorphism analyses to distinguish five species of Phytophthora associated with ornamental plants

Fatty acid methyl ester (FAME) profiles and amplified fragment length polymorphisms (AFLPs) were evaluated as tools for identifying species of Phytophthora. Five isolates of each of Phytophthora cactorum, Phytophthora citrophthora, Phytophthora cinnamomi, Phytophthora nicotianae and Phytophthora cryptogea were subjected to both analyses to examine variation among and within species. In FAME analysis, isolates of P. cactorum, P. cinnamomi and P.nicotianae were clustered by species, but isolates of P. citrophthora and P.cryptogea were divided into multiple clusters based on greater variations within these two species. The AFLP analysis differentiated all five species of Phytophthora. The five isolates of each species were grouped in a separate terminal cluster, but diversity within a species cluster varied considerably with variation greater in P. cryptogea and P. citrophthora. Comparing the dendrograms based on FAME and AFLP analyses, the overall patterns of both were similar. The P. cactorum cluster was distinct from clusters of the other four species, which formed one large cluster. The higher values of percentages of polymorphic loci and gene diversity in AFLP analysis substantiated diversity observed among isolates of P. citrophthora and P. cryptogea in FAME and AFLP dendrograms. Both FAME and AFLP appear to be useful tools for identifying species of Phytophthora, but only AFLP analysis has potential to study genetic and phylogenetic relationships within and among species in this genus.     

Electron microscope studies of oogonial wall and elemental composition of oogonia in Phytophthora

Scanning electron microscopy of oogonia of Phytophthora spp. showed that the oogonial wall was smooth in P. cactorum, P. citricola, P. heveae, and P. palmivora; finely granular in P. megasperma and P. megasperma var. sojae; and coarsely granular in P. parasitica. Transmission electron microscopy demonstrated that the oogonial wall in Phytophthora was composed of three layers with the middle layer being the least or the most electron dense. A coat of amorphous material was found on the entire outer surface of the oogonial wall. Elemental analysis of oogonia by means of a SEM electron probe microanalyzer revealed similar emission spectra among Phytophthora spp. with a characteristic peak for calcium.     

Phytophthora spp. from rubbertree plantations in Yunnan Province of China

Phytophthora spp. associated with leaf fall, stem canker and black stripe of rubbertree (Hevea brasiliensis Muell.-Arg.) in Xishuangbanna of Yunnan Province were isolated from leaves, bark, fruit and soil in the plantations. Of a total of 50 isolates, 42 were designated as P. citrophthora (R. E. Sm. & E. H. Sm.) Leonian, while the others were identified as P. palmivora (Butl.) Butl., P. colocasiae Rac. and P. cactorum (Leb. & Conn) Schroet. This appears to be the first report of P. citrophthora on Hevea brasiliensis.     

The Phytophthora diseases of stone fruit trees in Greece

Phytophthora collar and crown rots are serious soilborne diseases which for a long time have caused considerable losses in stone fruit orchards in Greece. A number of Phytophthora species are notorious for being the cause of crown and root rots in Greek stone trees, including P. cactorum, P. citricola, P. cryptogea, P. drechsleri, P. nicotianae, P. citrophthora, P. syringae and P. megasperma. The most important Phytophthora species is P. cactorum, while P. syringae and P. citrophthora may be locally significant. The economic consequences from death of peach trees and yield losses caused by this disease in Imathia County are serious.     

Survival and recovery of Phytophthora capsici and oomycetes in tailwater and soil from vegetable fields in Florida

A current trend in Florida agriculture to conserve water is to irrigate with surface runoff water (tailwater) recovered in retention ponds and canals. Water filtration and lemon leaf baiting recovered Phytophthora capsici and other plant pathogenic Oomycetes in runoff water from ponds and canals. A total of 196 isolates of Phytophthora spp. and 471 isolates of Pythium spp. were recovered. Phytophthora spp. included P. capsici, P. cinnamomi, P. lateralis, P. nicotianae, P. citricola, P. cryptogea and P. erythroseptica. Species of Pythium were P. aphanidermatum, P. catenulatum, P. helicoides, P. irregulare, P. myriotylum, and Pythium‘group F’. Isolates of P. aphanidermatum, P. irregulare, P. myriotylum, and Pythium‘group F’ were pathogenic on pepper and tomato. Recovery of P. capsici propagules was related to soil moisture‐holding capacity and time interval but not temperature. Recovery of P. capsici propagules at 100% soil moisture‐holding capacity and 30° C was 57 days. In tailwater, recovery of propagules of P. capsici was 63 days at 24°C to 25°C. The potential exists to reintroduce and disseminate species of Phytophthora and Pythium when using tailwater for irrigation or other practices.     

Occurrence of Pythium spp. and Phytophthora spp. in Norwegian Greenhouses and their Pathogenicity on Cucumber Seedlings

Samples of tomato, lettuce and cucumber submitted for diagnosis to the Plant Protection Centre at the Norwegian Crop Research Institute and samples of soil, water and cucumber collected from greenhouses employing hydroponic cultures were examined for the occurrence of Pythium spp. and Phytophthora spp. Two species of Phytophthora and 16 species of Pythium were identified. Phytophthora cryptogea was found on tomato and lettuce. Phytophthora nicotianae was found on tomato fruit. Phytophthora was not found on cucumbers. Pythium irregulare and Pythium group F were the two most commonly found Pythium species in hydroponically cultivated cucumbers. A pathogenicity test with 56 isolates was performed on cucumber seedlings. The most aggressive species were Pythium aphanidermatum, P. irregulare, Pythium paroecandrum and Pythium ultimum.     

Antagonism of Chaetomium globosum, Gliocladium virens and Trichoderma viride to four soil-borne Phytophthora species

Chaetomium globosum, Gliocladium virens and Trichoderma viride, isolated from soil samples from the Kivu-region of Zaire, are antagonistic and mycoparasitic to four soil-borne Phytophthora spp. Radial growth of Phytophthora cinnamomi, P. cactorum, P. fragariae or P. nicotianae mycelia was inhibited in all antagonist-pathogen combinations; mycelial mats of Phytophthora colonies were overgrown by the antagonists and hyphae lysed. Samples of mycoparasited mycelium, plated on benlateamended PDA to inhibit the development of antagonists, showed severely reduced vitality after 77 days of dual culture.     

Pathogenicity of four Phytophthora Species on Wild Cherry and Italian Alder Seedlings

Inoculation tests were carried out in the greenhouse on wild cherry (Prunus avium) and Italian alder (Alnus cordata) seedlings, to determine their susceptibility to certain Phytophthora species (P. citrophthora, P. alni, P. megasperma and P. cinnamomi) that are commonly present in the soil. Host susceptibility was evaluated in accordance with a disease index, with the lesion length after stem inoculation, and with a root system disease index. Wild cherry was found to be highly susceptible to P. citrophthora, and was also found to be susceptible to P. alni, although to a lesser extent. Italian alder was very susceptible to P. alni, but had only low susceptibility to P. citrophthora. The other Phytophthora species caused only modest symptoms. The danger to alder and wild cherry is all the greater because these trees not only share the same pathogens, but also commonly planted together in mixed stands. The results will now have to be confirmed by using a more natural inoculation method.     

Interactions between the soilborne root pathogenPhytophthora nicotianae var.parasitica and the arbuscular mycorrhizal fungusGlomus mosseae in tomato plants

In order to study the influence of Arbuscular Mycorrhiza (AM) on the development of root rot infection, tomato plants were raised with or withoutGlomus mosseae and/orPhytophthora nicotianae var.parasitica in a sand culture system. All plants were fed with a nutrient solution containing one of two phosphorus (P) levels, 32µM (I P) or 96µM (II P), to test the consequence of enhanced P nutrition by the AM fungus on disease dynamics. Mycorrhizal plants had a similar development to that of control plants. Treatment withPhytophthora nicotianae var.parasitica resulted in a visible reduction in plant weight and in a widespread root necrosis in plants without mycorrhiza. The presence of the AM fungus decreased both weight reduction and root necrosis. The percentage reduction of adventitious root necrosis and of necrotic root apices ranged between 63 and 89% The enhancement of P nutrition increased plant development, but did not appreciably decrease disease spread. In our system, mycorrhiza increased plant resistance toP. nicotianae var.parasitica infection. Although a contribution of P nutrition by mycorrhiza cannot be excluded, other mechanisms appear to play a crucial role.     

Effects of extracts of Alnus glutinosa seeds on growth of Frankia strain ArI3 under static and fermentor culture conditions

The amount of root mortality caused by root pathogens such as Phytophthora nicotianae (syn. Phytophthora parasitica) has typically been inferred from the net change in root length density in sequential soil cores. Because such measurements give information only on net changes in root populations, the actual rate of root turnover is often underestimated. We used minirhizotrons to track the fate of a large number of individual fine roots of mature field-grown citrus trees over a 6-month period. This method enabled us to examine the effect of P. nicotianae population levels on fine-root mortality. Seasonal and genotypic variation in patterns of citrus fine root mortality were associated with variation in population levels of P. nicotianae. Fine root lifespans were shorter when populations of P. nicotianae were high. Fine roots of the Phytophthora-susceptible rootstock, rough lemon (Citrus jamibhiri), had shorter median lifespans and supported larger populations of P. nicotianae than the fine roots of the more tolerant rootstock, Volkamer lemon (Citrus volkameriana). Rates of root mortality were either relatively constant for roots of all ages, or increased with age; the latter pattern was most pronounced for Volkamer lemon roots. Differences in the age-dependence of root mortality may, therefore, play a role in genotypic differences in tolerance of Phytophthora root rot by these two rootstocks. H Lambers Section editor     

Ultrastructure of Wall Swellings of Germinating Sporangia of Phytophthora palmivora (Butl.) Butl.

Germ tubes of directly germinating sporangia of P. palmivoraincubated in yeast extract solution at 30 ?C usually developedinto prominent swellings from which hyphae later emerged. Thegerm tubes arose as an extension of a new germination wall formedinternal to the sporangial wall prior to germination. The germtube swellings contained typical hyphal organelles. The germtube swelling possessed a thicker wall than both hyphae growingout of it and germ tubes that did not form swellings.     

Cloning,structural features,and expression analysis of resistance gene analogs in Tobacco

Using degenerate primers based on the conserved nucleotide binding site (NBS) and protein kinase domain (PKD), 100 resistance gene analogs (RGAs) were isolated from tobacco variety Nicotiana repanda. BLASTx search against the GenBank database revealed that 27 belong to the NBS class and 73 belong to the protein kinase (PK) class. Cluster analysis and multiple sequence alignment of the deduced protein sequences indicate that RGAs of the NBS class can be divided into two groups: toll/interleukin receptor (TIR) and non-TIR types. Both types possess 6 conserved motifs (P-loop, RNBS-A, Kinase-2, RNBS-B, RNBS-C, GLPL). Based on their sequence similarity, the tobacco RGAs of the PK class were assigned to 8 subclasses. We examined their expression after infection with either Tobacco mosaic virus (TMV) or the tobacco black shank pathogen (Phytophthora parasitica var. nicotianae). The expression levels of 4 RGAs of the PK class were significantly elevated by TMV and 1 RGA of the PK class and 3 RGAs of the NBS class were up-regulated by P. parasitica var. nicotianae. The expression of two RGAs of the PK class was induced by P. parasitica var. nicotianae. Infection by either TMV or P. parasitica var. nicotianae enhanced the expression of NtRGA2, a RGA of the PK class. The present study shows that RGAs are abundant in the tobacco genome and the identification of tobacco RGAs induced by pathogens should provide valuable information for cloning related resistance genes in tobacco.     

Timing and abundance of sporangia production and zoospore release influences the recovery of different Phytophthora species by baiting

Analysis of soil samples using High Throughput Sequencing (HTS) frequently detects more Phytophthora species compared with traditional soil baiting methods. This study investigated whether differences between species in the timing and abundance of sporangial production and zoospore release could be a reason for the lower number of species isolated by baiting. Stems of Eucalyptus marginata were inoculated with ten Phytophthora species (P. nicotianae, P. multivora, P. pseudocryptogea, P. cinnamomi, P. thermophila, P. arenaria, P. heveae, P. constricta, P. gondwanensis and P. versiformis), and lesioned sections for each species were baited separately in water. There were significant differences between species in timing of sporangia production and zoospore release. P. nicotianae, P. pseudocryptogea, P. multivora and P. thermophila released zoospores within 8–12 h and could be isolated from lesioned baits within 1–2 days. In contrast, P. constricta did not produce zoospores for over 48 h and was only isolated 5–7 days after baiting. P. heveae and P. versiformis did not produce zoospores and were not recovered from the baits. When species were paired in the same baiting tub, those that produced zoospores in the shortest time were isolated most frequently, while species slow to produce zoospores, or which produced them in lower numbers, were isolated from few baits or not at all. Thus, species differences in the timing of sporangia production and zoospore release may contribute to the ease of isolation of some Phytophthora species when they are present together with other Phytophthora species in an environmental sample.     

Phytophthora ×stagnum nothosp. nov., a New Hybrid from Irrigation Reservoirs at Ornamental Plant Nurseries in Virginia

A novel Phytophthora species was frequently recovered from irrigation reservoirs at several ornamental plant production facilities in eastern Virginia. Initial sequencing of the internal transcribed spacer (ITS) region of this species generated unreadable sequences due to continual polymorphic positions. Cloning and sequencing the ITS region as well as sequencing the mitochondrially encoded cytochrome c oxidase 1 and beta-tubulin genes revealed that it is a hybrid between P. taxon PgChlamydo as its paternal parent and an unknown species genetically close to P. mississippiae as its maternal parent. This hybrid has some diagnostic morphological features of P. taxon PgChlamydo and P. mississippiae. It produces catenulate hyphal swellings, characteristic of P. mississippiae, and chlamydospores, typical of P. taxon PgChlamydo. It also produces both ornamented and relatively smooth-walled oogonia. Ornamented oogonia are another important diagnostic character of P. mississippiae. The relatively smooth-walled oogonia may be indicative of oogonial character of P. taxon PgChlamydo. The new hybrid is described here as Phytophthora ×stagnum.