Helga Förster

Sequence analysis of the small subunit ribosomal RNAs of three zoosporic fungi and implications for fungal evolution.

ABSTRACTThe small-subunit ribosomal RNA gene sequences of the chytridiomycete Blastocladiella emersomi and the oomycetes Lagenidium giganteum and Phytophthora megasperma f. sp. glycinea were determ...

Effect of Phosphite on Tomato and Pepper Plants and on Susceptibility of Pepper to Phytophthora Root and Crown Rot in Hydroponic Culture

Tomato and pepper plants were grown hydroponically in a greenhouse using phosphate or technical and commercial formulations of phosphite as sources of phosphorus nutrition to determine the effects on plant development and susceptibility to Phytophthora root and crown rot. Phosphite-treated tomato and pepper plants were deficient of phosphate and developed phosphorus-deficiency symptoms. Growth of plants (leaf area and leaf, stem, and root dry weights) that were fertilized with phosphite was significantly (P < 0.05) reduced compared with phosphate-fertilized plants. In Phytophthora capsici-inoculated pepper plants, incidence of Phytophthora crown rot was significantly reduced in phosphite-treated plants compared with no phosphorus or phosphate-treated plants. Incidence of crown rot in pepper plants treated with 1 mM phosphate plus 0.3 mM phosphite was intermediate between plants treated with only phosphite (1 mM or 0.1 mM) and plants treated with phosphate (1 mM).

Comparative Efficacy of the New Postharvest Fungicides Azoxystrobin, Fludioxonil, and Pyrimethanil for Managing Citrus Green Mold

Three new fungicides (i.e., azoxystrobin, fludioxonil, and pyrimethanil) are currently being introduced for postharvest management of citrus green mold in the United States. The effectiveness of each fungicide was evaluated when applied alone (at 1,000 to 1,200 mg/liter) or in mixtures (at 500 mg/liter each component) to lemon fruit that were wound-inoculated with imazalil/thiabendazole (TBZ)-sensitive or -resistant isolates of Penicillium digitatum. In laboratory studies when aqueous fungicide solutions were applied 9 to 21 h after inoculation, pyrimethanil showed the highest level of green mold control. The efficacy of fludioxonil and azoxystrobin was very high at the early timings, but decreased as time after inoculation increased. Differences in fungicide performance were not due to multiple fungicide resistance, but more likely due to differences in fungicide mobility in fruit tissue. Azoxystrobin-fludioxonil mixtures were significantly more effective when compared to single-fungicide treatments. Mixtures of imazalil with pyrimethanil were the most effective in controlling decay. The efficacy of all fungicides was significantly lower when mixed into a packing fruit coating as compared to aqueous or storage fruit coating applications. In laboratory and packingline studies, the lowest incidence of green mold decay was obtained when azoxystrobin-fludioxonil and imazalil-pyrimethanil were applied as aqueous solutions that were followed by a fruit coating. Among the new fungicides, azoxystrobin and fludioxonil applied in water or storage fruit coating, respectively, provided the best anti-sporulation activity. Storage fruit coating improved the activity of both fungicides. Pyrimethanil was the least effective fungicide in suppressing sporulation of the pathogen on decaying fruit. Overall, among the mixtures, azoxystrobin-fludioxonil and TBZ-fludioxonil had high anti-sporulation activity in aqueous and storage fruit coating applications. New integrated management programs should be based on monitoring of fungicide sensitivities in pathogen populations, rotating mixtures of products with different modes of action, and using appropriate fungicide application strategies.

Phylogenetic relationships of Phytophthora species based on ribosomal ITS I DNA sequence analysis with emphasis on Waterhouse groups V and VI

Phylogenetic relationships among Phytophthora species were investigated by sequence analysis of the internal transcribed spacer region I of the ribosomal DNA repeat unit. The extensive collection of isolates included taxa from all six morphological groups recognized by Waterhouse (1963) including molecular groups previously identified using isozymes and mtDNA restriction fragment length polymorphisms. Similar to previous studies, the inferred relationships indicated that molecular groups of P. cryptogea}drechslerilike and P. megasperma-like taxa are polyphyletic. Morphological groups V and VI, which are dierentiated by the presence of amphigynous or paragynous antheridia, are not monophyletic: species of the two groups are interspersed in the tree. Species with papillate and semi-papillate sporangia (groups I‐IV) clustered together and this cluster was distinct from those of species with nonpapillate sporangia. There was no congruence between the mode of antheridial attachment, sporangial caducity, or homo- or heterothallic habit and the molecular grouping of the species. Our study provides evidence that the antheridial position together with homo- or heterothallic habit does not reflect phylogenetic relationships within Phytophthora. Consequently, confirming studies done previously (Cooke & Duncan 1997), this study provides evidence that the morphological characters used in Phytophthora taxonomy are of limited value for deducing phylogenetic relationships, because they exhibit convergent evolution.

Taxonomic structure of Phytophthora cryptogea and P. drechsleri based on isozyme and mitochondrial DNA analyses

Intra- and interspecific isozyme variation was evaluated for 123 isolates assigned to either Phytophthora cryptogea or P. drechsleri , and compared with that of 15 isolates of P. erythroseptica and 11 isolates of P. lateralis . Isolates of P. cryptogea and P. drechsleri were from worldwide sources and displayed a high degree of variability. The majority of these isolates were subsequently divided into ten distinct groups based on numerical analysis of 24 putative enzyme loci. None of the enzyme loci were monomorphic for all ten groups. Analysis of mitochondrial (mt) DNA restriction fragment length polymorphisms of selected isolates from each isozyme group supported the isozyme data. Differences in morphological features of the ten isozyme groups of P. cryptogea and P. drechsleri were not sufficiently distinct to readily distinguish between them. Isozyme analysis of P. erythroseptica revealed that it is a uniform and distinct taxon. The isolates of P. lateralis also formed a homogeneous and discrete group. An interspecific comparison revealed that the variation among the ten isozyme groups of P. cryptogea and P. drechsleri was as great as that observed among P. cinnamomi, P. cambivora, P. lateralis, P. erythroseptica and P. richardiae . The combined results of isozyme and mtDNA analysis indicate that there are at least seven distinct molecular species represented by the 123 isolates of P. cryptogea and P. drechsleri evaluated in this study.

Characterization of genetic and biochemical mechanisms of fludioxonil and pyrimethanil resistance in field isolates of Penicillium digitatum.

Genetic and biochemical mechanisms of fludioxonil and pyrimethanil resistance in isolates of Penicillium digitatum were evaluated and compared to those characterized in other fungi. Resistant isolates were naturally occurring in packinghouses and were not associated with crop losses. For the phenylpyrrole fludioxonil, EC50 values were 0.02 to 0.04 microg/ml for sensitive, 0.08 to 0.65 microg/ml for moderately resistant (MR), and > 40 microg/ml for highly resistant (HR) isolates. Two fludioxonil-sensitive isolates evaluated were also significantly more sensitive to the unrelated dicarboximide fungicide iprodione, that also disrupts osmotic regulation, than the MR and HR isolates. There was no consistent relationship, however, between the HR and MR isolates and their sensitivity to iprodione or osmotic stress. Although, two nucleotide substitutions were found in a sequence analysis of the N-terminal amino acid repeat region of the os-1-related histidine kinase gene among isolates of P. digitatum, these were not correlated with fludioxonil resistance. In mycelia not exposed to fludioxonil, the amount of phosphorylated OS-2-related protein (PdOS-2) was higher in fludioxonil-sensitive isolates and lowest in the HR isolate. An increase in PdOS-2 was observed for sensitive and resistant isolates after exposure to fludioxonil. In addition, glycerol content in untreated mycelia of the fludioxonil-sensitive isolate was significantly higher than in resistant isolates. After exposure to fludioxonil, glycerol concentrations significantly increased in the sensitive and MR isolates, but not in the HR isolate. Thus, our studies indicate that the mode of action of fludioxonil in P. digitatum is probably the mitogen-activated protein kinase pathway that stimulates glycerol synthesis in sensitive and MR isolates. The general suppression of this pathway in resistant isolates was supported by the fact that growth and sporulation of MR and HR isolates were significantly reduced from that of sensitive isolates. In studies on the mode of action of anilinopyrimidines (AP), EC50 values for mycelial growth of P. digitatum and the previously characterized Botrytis cinerea were determined for cyprodinil and pyrimethanil using a defined culture medium without and with the addition of selected amino acids and homocysteine. The addition of amino acids resulted in a reduced toxicity of the two AP fungicides in both fungi, but the effect of each additive was significantly lower for P. digitatum than for B. cinerea. This suggests that methionine biosynthesis is not the primary target site of APs in P. digitatum.

Molecular taxonomy of Phytophthora megasperma based on mitochondrial and nuclear DNA polymorphisms.

One hundred and ninety-four isolates of P. megasperma from a wide range of host plants and of worldwide origin were subjected to molecular characterization based on numerical analyses of their mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs). Of the nine distinct molecular groups identified, four represented groups of isolates from single host plants. In addition to the previously recognized host-specific taxa isolated from alfalfa, clover and soybean, a previously unidentified, fourth group contained only isolates from asparagus. An additional mtDNA group included isolates recovered primarily from Douglas fir. Most of the remaining isolates, from a wide range of woody and herbaceous hosts, were placed into another four groups. Of these, groups A, B and C were closely related; group D was not. A number of additional isolates had unique mtDNA patterns. An intergroup comparison confirmed the genetic distinctness of the nine groups and intergroup genetic distances were sometimes large. In addition, isolates representing specific molecular groups of the P. cryptogea/P. drechsleri ‘species complex’ ( Mills, Forster & Coffey, 1991 ) were interspersed between those of the P. megasperma groups. The mtDNA RFLP findings were largely supported by an analysis based on chromosomal DNA RFLPs. However, amongst the three mtDNA groups A, B and C containing isolates from a broad host range (BHR protein group; Hansen et al., 1986 ), only group A was resolved.

Spiral gradient dilution, a rapid method for determining growth responses and 50% effective concentration values in fungus-fungicide interactions.

ABSTRACT A new technique, the spiral gradient dilution method, was evaluated for determining 50% effective concentrations (EC(50) values) of fungicides for the inhibition of mycelial growth and conidial germination of various fungi. In this method, an agar medium is plated with a fungicide solution by means of a spiral plater, which applies the fungicide in a 2.5-log dilution in a continuous radial concentration gradient. Fungal inoculum is then placed along radial lines across the gradient. After incubation of the plates, distinct growth shapes were observed in different fungus-fungicide interactions. Mycelial growth responses to increasing fungicide concentrations ranged from abrupt to gradual transitions. Conidial germination responses were similar; in addition, distinct zones of confluent growth, nonconfluent growth, and outlier colonies were also identified, depending on the fungus-fungicide interaction. The fungicide concentration at the radial distance at which 50% reduction of growth or spore germination occurred, compared with growth or germination on unamended media, was calculated by a computer program. EC(50) values were obtained for mycelial growth in 22 fungus-fungicide interactions and for conidial germination in five interactions. The fungi evaluated were members of the Zygomycota, Ascomycota, Basidiomycota, and Deuteromycota. Nine fungicides, belonging to six different chemical classes, were tested. EC(50) values were compared with those obtained by the traditional agar dilution method. In linear regression analyses of the two methods, the models were highly significant (P < 0.01), and coefficients of determination (r(2)) were 0.92 for the mycelial growth assays and 0.94 for the conidial germination assays. Regression slopes were not significantly different from 1 (P > 0.05) with optimal program settings in the software. Estimated bias, coefficients of variation, and actual confidence intervals for the regression slope were 13.5%, 6.5%, and 1.14 +/- 0.14 for the mycelial growth assays and 7.5%, 14.5%, and 1.08 +/- 0.37 for the conidial germination assays. These analyses indicate that the spiral gradient dilution method is accurate and precise compared with the agar dilution method for calculating EC(50) values of fungicides in continuous growth responses to fungicide concentration gradients.

Evidence for distinct isozyme subgroups within Phytophthora citricola and close relationships with P. capsici and P. citrophthora.

Fourteen isozyme loci resolved ten electrophoretic types (ETs) amongst a worldwide collection of 125 isolates of P. citricola . The ETs clustered into five distinct subgroups, CIT1–5. CIT1 contained all the isolates from Citrus spp. including the Sawada type culture, the now defunct species P. pini and an authentic isolate of P. cactorum var. applanata . Isolates in subgroups CIT1, CIT2, and CIT3, which differed from each other at only a few enzyme loci, were from diverse locations and hosts. Subgroups CIT4 and CIT5 differed from one another at eight loci and from the other three subgroups by four and five loci, respectively. CIT4 was composed of isolates from native South African plants, and CIT5 of isolates from avocado only. Two isolates from Psidium guajava , previously identified as P. citricola (Ko, 1983), were only distantly related to CIT1–5. Three enzymes (phosphoglucose isomerase, malate dehydrogenase and menadione nitrate reductase) could be used to define subgroups. Examination of temperature growth responses as well as sporangial and oogonial morphology revealed no difference among isolates from subgroups CIT1, CIT2 and CIT3. In contrast, isolates of the avocado subgroup, CIT5, did not survive at temperatures at or above 30 °C, whilst isolates of subgroups CIT1–3 survived temperatures up to 33°. In addition, isolates of CIT5 produced smaller oogonia than those from CIT1–3, although there was some overlap. A comparison of the P. citricola subgroups with P. cactorum revealed a high dissimilarity based on isozyme analysis. However, a close relationship was established between P. citricola, P. capsici and P. citrophthora from both isozyme analysis and mitochondrial DNA restriction patterns. The host-specific group CIT5 clustered with P. capsici and P. citrophthora rather than with other P. citricola subgroups, and may be sufficiently genetically distinct from other subgroups of P. citricola to be regarded as a separate biological taxon.

Optimizing Efficacy of New Postharvest Fungicides and Evaluation of Sanitizing Agents for Managing Citrus Green Mold

Three new fungicides, azoxystrobin, fludioxonil, and pyrimethanil, that belong to different chemical classes are highly effective in managing citrus green mold and are being registered for postharvest use in the United States. Recirculating in-line drenches provided a significantly improved efficacy compared with standard low-volume spray applications. To prevent pathogen contamination of drench solutions, two oxidizing disinfectants, sodium hypochlorite and hydrogen peroxide/peroxyacetic acid (HPPA) solutions, were evaluated. Inhibition of conidial germination of Penicillium digitatum was dependent on the pH of the solution and the exposure time for each sanitizing agent. Chlorine (50 mg/liter) and HPPA (2,700 mg/liter) effectively inhibited germination in 40- and 240-s exposures, respectively, at pH 7. All fungicides tested were compatible and effective with HPPA, whereas fludioxonil, azoxystrobin, and thiabendazole, but not imazalil and pyrimethanil, were compatible with chlorine. In laboratory studies, sodium bicarbonate (SBC, 3%) significantly increased the efficacy of the three fungicides (250 mg/liter) and had no adverse effect on their stability in aqueous solutions. Fludioxonil (300 mg/liter)-SBC mixtures were still highly effective when applied 24 h after fruit inoculation. In experimental packingline studies, SBC or SBC-chlorine improved the efficacy of fludioxonil, whereas azoxystrobin was effective with and without these additives. Heating of drench solutions of fludioxonil (300 mg/liter) to 50°C did not improve decay control. In conclusion, in-line recirculating drench applications and fungicide-sanitizer-SBC mixtures significantly increased fungicide efficacy and provide an integrated approach for optimizing fungicide efficacy. These strategies also should minimize the selection for resistant isolates of the pathogen.