New and Interesting Fungi. 3.

Seven new genera, 26 new species, 10 new combinations, two epitypes, one new name, and 20 interesting new host and / or geographical records are introduced in this study. New genera are: Italiofungus (based on Italiofungus phillyreae) on leaves of Phillyrea latifolia (Italy); Neolamproconium (based on Neolamproconium silvestre) on branch of Tilia sp. (Ukraine); Neosorocybe (based on Neosorocybe pini) on trunk of Pinus sylvestris (Ukraine); Nothoseptoria (based on Nothoseptoria caraganae) on leaves of Caragana arborescens (Russia); Pruniphilomyces (based on Pruniphilomyces circumscissus) on Prunus cerasus (Russia); Vesiculozygosporium (based on Vesiculozygosporium echinosporum) on leaves of Muntingia calabura (Malaysia); Longiseptatispora (based on Longiseptatispora curvata) on leaves of Lonicera tatarica (Russia). New species are: Barrmaelia serenoae on leaf of Serenoa repens (USA); Chaetopsina gautengina on leaves of unidentified grass (South Africa); Chloridium pini on fallen trunk of Pinus sylvestris (Ukraine); Cadophora fallopiae on stems of Reynoutria sachalinensis (Poland); Coleophoma eucalyptigena on leaf litter of Eucalyptus sp. (Spain); Cylindrium corymbiae on leaves of Corymbia maculata (Australia); Diaporthe tarchonanthi on leaves of Tarchonanthus littoralis (South Africa); Elsinoe eucalyptorum on leaves of Eucalyptus propinqua (Australia); Exophiala quercina on dead wood of Quercus sp., (Germany); Fusarium californicum on cambium of budwood of Prunus dulcis (USA); Hypomyces gamsii on wood of Alnus glutinosa (Ukraine); Kalmusia araucariae on leaves of Araucaria bidwillii (USA); Lectera sambuci on leaves of Sambucus nigra (Russia); Melanomma populicola on fallen twig of Populus canadensis (Netherlands), Neocladosporium syringae on branches of Syringa vulgarishorus (Ukraine); Paraconiothyrium iridis on leaves of Iris pseudacorus (Ukraine); Pararoussoella quercina on branch of Quercus robur (Ukraine); Phialemonium pulveris from bore dust of deathwatch beetle (France); Polyscytalum pinicola on needles of Pinus tecunumanii (Malaysia); Acervuloseptoria fraxini on Fraxinus pennsylvanica (Russia); Roussoella arundinacea on culms of Arundo donax (Spain); Sphaerulina neoaceris on leaves of Acer negundo (Russia); Sphaerulina salicicola on leaves of Salix fragilis (Russia); Trichomerium syzygii on leaves of Syzygium cordatum (South Africa); Uzbekistanica vitis-viniferae on dead stem of Vitis vinifera (Ukraine); Vermiculariopsiella eucalyptigena on leaves of Eucalyptus sp. (Australia).

First Report of Geosmithia morbida on English Walnut and its Paradox Rootstock in California.

Geosmithia morbida, the causal agent of thousand cankers disease (TCD), is vectored by the walnut twig beetle (WTB), Pityophthorus juglandis, causing decline in eastern black walnut, Juglans nigra (4), and canker development on many Juglans species (5). In the summer of 2012, a survey for TCD incidence in English walnut, J. regia, in orchards in California identified many trees with WTB activity and characteristic TCD symptoms. Both the J. regia scion and its Paradox hybrid rootstock (J. hindsii× J. regia) were affected. In some cases, trees exhibited bleeding on the bark surface from WTB entrance holes. Removal of the outer bark revealed cankers in the phloem around the WTB galleries. Two samples were taken from scions and three samples were collected from rootstocks of trees in orchards in northern California. Pieces (~3 to 4 mm2) of symptomatic tissue were placed in acidified potato dextrose agar (APDA), and the plates were incubated for 4 to 5 days at 30°C. Samples exhibiting fungal growth similar in morphology to G. morbida were transferred to PDA plates to obtain pure cultures and then processed to obtain single-spore cultures. Culture morphology for five single-spore isolates (Gm103, Gm104, Gm105, Gm107, and Gm108) was similar to that described by Kolarík et al. (4) for G. morbida. Conidiophores were penicillate and verrucose. Conidia were narrowly cylindrical, 5.2 ± 0.06 × 2.2 ± 0.04 µm (n = 50). Single-spore isolates were then grown in 1% yeast extract glucose liquid culture for 7 to 10 days. DNA was extracted and the ITS region was amplified, including the 5.8S region by using primers ITS1F/ITS4. Sequences were assembled and deposited in GenBank under accessions KJ664793 to KJ664797. Sequences were compared to those in GenBank; all sequences matched (99 to 100% identity) the ITS sequences of G. morbida strain CBS 124663. Pathogenicity tests were performed on 28-cm-long detached branches of J. regia. Four branches per isolate were inoculated with a 5-mm-diameter mycelial plug from a 2-week-old culture. Branches were incubated at room temperature (23 ± 2°C) in a humidified container for 3 weeks, and then canker lengths were measured. Pieces of the cankered area were placed in APDA and incubated as described above with G. morbida re-isolated from the cankers for all of the isolates, completing Koch's postulates. Average canker lengths ranged from 48.6 ± 4.3 to 72.1 ± 7.1 mm. Re-isolated G. morbida exhibited the same growth and reproductive structure morphology in culture on PDA as the original cultures. TCD in association with WTB has been observed in California English walnut orchards since 2008 (1,2,3). However, this is the first report for completion of Koch's postulates and morphological and molecular confirmation of G. morbida in J. regia and the Paradox rootstock, the predominant rootstock used in commercial orchards. TCD is a concern to the walnut industry in California with over 245,000 bearing acres reported in 2012. References: (1) M. Flint et al. CAPCA Adviser 8:36, 2010. (2) A. D. Graves et al. Walnut Twig Beetle and Thousand Cankers Disease: Field Identification Guide. UC-IPM website publication, http://www.ipm.ucdavis.edu/PDF/MISC/thousand_cankers_field_guide.pdf , 2009. (3) J. Hasey et al. (Abstr.) Phytopathology 100:S48, 2010. (4) M. Kolarík et al. Mycologia 103:325, 2011. (5) C. Utley et al. Plant Dis. 97:601, 2013.

Phytophthora ramorum does not cause physiologically significant systemic injury to California bay laurel, its primary reservoir host.

California bay laurel trees (Umbellularia californica) play a crucial role in the reproduction and survival of Phytophthora ramorum in coastal California forests by supporting sporulation during the rainy season and by providing a means for the pathogen to survive the dry, Mediterranean summer. While bay laurel is thus critical to the epidemiology of sudden oak death and other P. ramorum diseases in California, the relatively minor symptoms observed on this reservoir host suggest that it may not sustain ecologically significant injury itself. The long-term role that P. ramorum will play in California forests will depend in part on the extent to which this pathogen decreases the ecological fitness of bay laurel. Despite the importance of this question, no study has yet investigated in detail the physiological impact that ramorum blight imposes on bay laurel. This experimental study quantifies the impact that P. ramorum has on artificially inoculated bay laurel seedlings with measurements that integrate the full injury that infection with an oomycete may cause: photosynthetic efficiency, total photosynthetic area, and growth. Leaf area and leaf mass were not impacted significantly by infection of P. ramorum. Photosynthetic efficiency was mildly depressed in symptomatic, but not asymptomatic leaves, despite unnaturally high levels of necrosis that were imposed on the seedlings. These results demonstrate that bay laurel trees suffer only minor injury from ramorum blight beyond visible necrotic symptoms. Consequently, it is highly likely that bay laurel will continue to be widely available as a host for P. ramorum in California forests, which has long-term implications for the composition of these forests.

Affinity purification and characterization of a cutinase from the fungal plant pathogen Monilinia fructicola (Wint.) honey.

Trifluoromethyl ketones (TFK) are potent inhibitors of a variety of serine hydrolases. The TFK inhibitor, 3-(4-mercaptobutylthio)-1,1,1-trifluoro-2-propanone (MBTFP), was found to competitively inhibit cutinase activity (I50 = 9.4 x 10(-3)) from the fungal plant pathogen Monilinia fructicola and to serve as an effective affinity ligand for the purification of cutinases from culture filtrates. The TFK inhibitors, 3-n-octylthio-1,1,1-trifluoro-2-propanone (OTFP) and 3-n-pentylthio-1,1,1-trifluoro-2-propanone (PTFP), also inhibited cutinase activity with I50 values of 1.6 x 10(-6) and 2.3 x 10(-4) M, respectively. Buffer containing OTFP was the strongest eluant for cutinases of M. fructicola and provided the best purification factor and yield, although buffers containing OTFP, detergent, and salt were found to be effective for eluting cutinases bound to MBTFP-Sepharose. Buffer containing 0.5% Triton X-100 also selectively eluted cutinases from the affinity column. Two-dimensional electrophoretic analysis by SDS-PAGE and isoelectric focusing of the affinity-purified cutinase fraction indicated activity associated with proteins of pI 8.2 and molecular masses of approximately 18.6 and 20.8 kDa. These proteins hydrolyzed [3H]cutin and artificial substrates such as p-nitrophenylbutyrate and related esters, typical of other cutinases, but differ from previously characterized cutinases in molecular mass. The two low-molecular-weight proteins resolved by 2-D gel electrophoresis were subjected to in-gel digestion with Lys-C and the resulting peptide fragments were separated by Microbore-HPLC. The amino acid sequences of several internal peptide fragments had high homology with cutinase sequences from other fungi, particularly the plant pathogen Botrytis cinerea. Our study illustrates the potential of TFK ligands for the affinity purification of cutinases and indicates that the cutinases from M. fructicola have novel features warranting further study.

Influence of stem diameter, water content, and freezing-thawing on bacterial canker development in excised stems of dormant stone fruit.

ABSTRACT In excised dormant stems of peach (Prunus persica), prune (Prunus domestica), and almond (Prunus dulcis), stem diameter, stem hydration, and freezing-thawing influenced the extent of infection caused by Pseudomonas syringae pv. syringae. Bacterial lesion length increased with increasing stem diameter, demonstrating the need to account for the effects of stem diameter when lesion length data are analyzed. Lesion length increased or decreased with stem hydration or dehydration, respectively. However, tissue water content was not a good indicator of tissue susceptibility to infection by P. syringae pv. syringae, as larger diameter stems had larger lesions and lower water content than did smaller diameter stems. After freezing at -5 degrees C for 12 to 24 h, inoculations made during the thawing process produced significantly larger lesions than did inoculations performed before freezing or after thawing. These results support the hypothesis that the increased susceptibility to bacterial canker that is associated with noninjurious freezing is a result of the increased passive spread of bacteria through water redistribution when inoculation is performed during the thawing process. Plant tissue water relationship characteristics that can influence water movement during freezing and thawing may be an important component of bacterial canker development in stone fruit trees.

Improved Detection of Polygalacturonase Activity due to Mucor piriformis with a Modified Dinitrosalicylic Acid Reagent.

ABSTRACT An assay for determination of galacturonic acid with 3,5-dinitrosalicylic acid was developed that substantially extends the linear range of detection compared to a previously published method with this reagent. In the improved assay, galacturonic acid was detected with a reagent containing 44 mM 3,5-dinitrosalicylic acid, 4 mM sodium sulfite, and 375 mM sodium hydroxide. The absorbance of the solution after reaction with galacturonic acid was determined at 575 nm and was linear at concentrations of galacturonic acid up to 50 mumol, with a lower limit of detection at ~400 nmol. The assay with the improved reagent could be performed in wavelength ranges from 550 to 575 nm, with higher sensitivity at the shorter wavelengths. The new reagent was used in routine assays of polygalacturonase activity in culture filtrates of the important postharvest fungal pathogen Mucor piriformis.

Apoptosis: A Functional Paradigm for Programmed Plant Cell Death Induced by a Host-Selective Phytotoxin and Invoked during Development.

The host-selective AAL toxins secreted by Alternaria alternata f sp lycopersici are primary chemical determinants in the Alternaria stem canker disease of tomato. The AAL toxins are members of a new class of sphinganine analog mycotoxins that cause cell death in both animals and plants. Here, we report detection of stereotypic hallmarks of apoptosis during cell death induced by these toxins in tomato. DNA ladders were observed during cell death in toxin-treated tomato protoplasts and leaflets. The intensity of the DNA ladders was enhanced by Ca2+ and inhibited by Zn2+. The progressive delineation of fragmented DNA into distinct bodies, coincident with the appearance of DNA ladders, also was observed during death of toxin-treated tomato protoplasts. In situ analysis of cells dying during development in both onion root caps and tomato leaf tracheary elements revealed DNA fragmentation localized to the dying cells as well as the additional formation of apoptotic-like bodies in sloughing root cap cells. We conclude that the fundamental elements of apoptosis, as characterized in animals, are conserved in plants. The apoptotic process may be expressed during some developmental transitions and is the functional process by which symptomatic lesions are formed in the Alternaria stem canker disease of tomato. Sphinganine analog mycotoxins may be used to characterize further signaling pathways leading to apoptosis in plants.

In situ isolation of mRNA from individual plant cells: creation of cell-specific cDNA libraries.

A method for isolating and cloning mRNA populations from individual cells in living, intact plant tissues is described. The contents of individual cells were aspirated into micropipette tips filled with RNA extraction buffer. The mRNA from these cells was purified by binding to oligo(dT)-linked magnetic beads and amplified on the beads using reverse transcription and PCR. The cell-specific nature of the isolated mRNA was verified by creating cDNA libraries from individual tomato leaf epidermal and guard cell mRNA preparations. In testing the reproducibility of the method, we discovered an inherent limitation of PCR amplification from small amounts of any complex template. This phenomenon, which we have termed the "Monte Carlo" effect, is created by small and random differences in amplification efficiency between individual templates in an amplifying cDNA population. The Monte Carlo effect is dependent upon template concentration: the lower the abundance of any template, the less likely its true abundance will be reflected in the amplified library. Quantitative assessment of the Monte Carlo effect revealed that only rare mRNAs (< or = 0.04% of polyadenylylated mRNA) exhibited significant variation in amplification at the single-cell level. The cDNA cloning approach we describe should be useful for a broad range of cell-specific biological applications.

Involvement of de Novo Protein Synthesis, Protein Kinase, Extracellular Ca2+, and Lipoxygenase in Arachidonic Acid Induction of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Genes and Isoprenoid Accumulation in Potato (Solanum tuberosum L.).

A series of inhibitors were tested to determine the participation of de novo protein synthesis, protein kinase activity, extracellular Ca2+, and lipoxygenase activity in arachidonic acid elicitation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene expression and sesquiterpene phytoalexin biosynthesis in potato (Solanum tuberosum L. cv Kennebec). Gene-specific probes were used to discriminate effects on the expression of two HMGR genes (hmg1 and hmg2) that respond differentially in tuber tissue following wounding or elicitor treatment. Inhibition of protein synthesis with cycloheximide completely blocked arachidonate-induced hypersensitive necrosis and browning, including HMGR gene induction and phytoalexin accumulation. This suggests that proteins necessary for coupling arachidonic acid reception to HMGR mRNA accumulation are either rapidly turned over or not present constitutively and are induced following elicitor treatment. Staurosporin, a potent inhibitor of protein kinases, and ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime]-tetraacetic acid, a Ca2+ chelator, inhibited arachidonate-induction of hmg2 gene expression and phytoalexin accumulation but did not inhibit the wound-induced expression of hmg1. However, staurosporin inhibited arachidonate's suppression of hmg1 gene expression. Eicosatetraynoic acid, a lipoxygenase inhibitor that suppresses elicitor-induced phytoalexin accumulation, also inhibited arachidonate's suppression of hmg1 and induction of hmg2. The results indicate that arachidonate's suppression of hmg1 and activation of hmg2 depend on a common intermediate or set of intermediates whose generation is sensitive to the inhibitors tested.

Lipid-derived signals that discriminate wound- and pathogen-responsive isoprenoid pathways in plants: methyl jasmonate and the fungal elicitor arachidonic acid induce different 3-hydroxy-3-methylglutaryl-coenzyme A reductase genes and antimicrobial isoprenoids in Solanum tuberosum L.

Induction of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR; EC 1.1.1.34) is essential for the synthesis of steroid derivatives and sesquiterpenoid phytoalexins in solanaceous plants following mechanical injury or pathogen infection. Gene-specific probes corresponding to different HMGR genes (hmg1 and hmg2) were used to study HMGR expression in potato tissue following treatment with methyl jasmonate, a lipoxygenase product of linolenic acid, or arachidonic acid, an elicitor present in the lipids of the potato late blight fungus Phytophthora infestans. Treatment of potato discs (2.2 cm in diameter) with low concentrations (0.45-45 nmol per disc surface) of methyl jasmonate nearly doubled the wound-induced accumulation of hmg1 transcripts and steroid-glycoalkaloid (SGA) accumulation, reduced the abundance of hmg2 transcripts, and did not induce phytoalexins. High concentrations of methyl jasmonate (2-4.5 mol per disc surface) suppressed hmg1 mRNA and SGA accumulation but did not affect hmg2 mRNA abundance or induce phytoalexins. In contrast, arachidonate treatment strongly suppressed hmg1 and strongly induced hmg2 mRNA in a concentration-dependent manner. There was a corresponding suppression of SGA accumulation and an induction of sesquiterpene phytoalexin accumulation by this elicitor. Lipoxygenase inhibitors reduced the wound-induced accumulation of hmg1 transcripts and suppressed SGA levels, effects that were overcome by exogenous methyl jasmonate (45 nmol per disc surface). The results (i) suggest that methyl jasmonate can function as a signal for hmg1 expression and SGA induction following wounding and (ii) indicate that the arachidonate- and jasmonate-response pathways are distinct in relation to HMGR gene expression and isoprenoid product accumulation. The results also are consistent with placement of the HMGR activities encoded by hmg1 and hmg2 within discrete steroid and sesquiterpenoid biosynthetic channels.

The 73-kb pIAA plasmid increases competitive fitness of Pseudomonas syringae subspecies savastanoi in oleander.

Pseudomonas syringae subsp. savastanoi causes tumors on olive and oleander by producing the plant growth regulators indoleacetic acid (IAA) and cytokinins following infection of the plant. The contribution of IAA production to the ability of P. syringae subsp. savastanoi to grow and survive in oleander leaf tissue was studied. Bacterial strains differing only with respect to IAA production were characterized. Growth and survival of wild-type and two mutant strains of P. syringae subsp. savastanoi in oleander leaf tissue were monitored by weekly colony counts and IAA plate assays. Growth rate of the three strains in culture and in planta did not differ significantly. However, the wild-type strain reached a higher population density and maintained its maximum density at least 9 weeks longer than either mutant population. An insertion mutant containing the IAA plasmid (pIAA), but incapable of IAA production, did not maintain a higher population density than a strain cured of the IAA plasmid. The pIAA-cured strain maintained a higher population density when coinoculated with an IAA-producing strain than when inoculated alone. These results suggest that IAA production may contribute to the fitness of P. syringae subsp. savastanoi in oleander tissue and that the iaa operon alone may be responsible for the competitive advantage of cells harboring pIAA.

Rapid stimulation of 5-lipoxygenase activity in potato by the fungal elicitor arachidonic Acid.

The activity of lipoxygenase (LOX) in aged potato tuber discs increased by almost 2-fold following treatment of the discs with the fungal elicitor arachidonic acid (AA). Enzyme activity increased above that in untreated discs within 30 min after AA treatment, peaked at 1 to 3 h, and returned to near control levels by 6 h. The majority of the activity was detected in a soluble fraction (105,000g supernatant), but a minor portion was also associated with a particulate fraction enriched in microsomal membranes (105,000g pellet); both activities were similarly induced. 5-Hydroperoxyeicosatetraenoic acid was the principal product following incubation of these extracts with AA. Antibodies to soybean LOX strongly reacted with a protein with a molecular mass of approximately 95-kD present in both soluble and particulate fractions whose abundance generally corresponded with LOX activity in extracts. LOX activity was not enhanced by treatment of the discs with nonelicitor fatty acids or by branched beta-glucans from the mycelium of Phytophthora infestans. Prior treatment of the discs with abscisic acid, salicylhydroxamic acid, or n-propyl gallate, all of which have been shown to suppress AA induction of the hypersensitive response, inhibited the AA-induced increment in LOX activity. Cycloheximide pretreatment, which abolishes AA elicitor activity for other responses such as phytoalexin induction, did not inhibit LOX activity in water- or elicitor-treated discs but enhanced activity similar to that observed by AA treatment. The elicitor-induced increase in 5-LOX activity preceded or temporally paralleled the induction of other studied responses to AA, including the accumulation of mRNAs for 3-hydroxy-3-methylglutaryl coenzyme A reductase and phenylalanine ammonia lyase reported here. The results are discussed in relation to the proposed role of the 5-LOX in signal-response coupling of arachidonate elicitation of the hypersensitive response.

Differential induction and suppression of potato 3-hydroxy-3-methylglutaryl coenzyme A reductase genes in response to Phytophthora infestans and to its elicitor arachidonic acid.

Induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is essential for the biosynthesis of sesquiterpenoid phytoalexins and steroid derivatives in Solanaceous plants following stresses imposed by wounding and pathogen infection. To better understand this complex step in stress-responsive isoprenoid synthesis, we isolated three classes of cDNAS encoding HMGR (hmg1, hmg2, and hmg3) from a potato tuber library using a probe derived from an Arabidopsis HMGR cDNA. The potato cDNAs had extensive homology in portions of the protein coding regions but had low homology in the 3' untranslated regions. RNA gel blot analyses using gene-specific probes showed that hmg1 was strongly induced in tuber tissue by wounding, but the wound induction was strongly suppressed by treatment of the tissue with the fungal elicitor arachidonic acid or by inoculation with an incompatible or compatible race of the fungal pathogen Phytophtora infestans. The hmg2 and hmg3 mRNAs also accumulated in response to wounding, but in contrast to hmg1, these mRNAs were strongly enhanced by arachidonic acid or inoculation. Inoculation with a compatible race of P. infestans resulted in similar patterns in HMGR gene expression of hmg2 and hmg3 except that the magnitude and rate of the changes in mRNA levels were reduced relative to the incompatible interaction. The differential regulation of members of the HMGR gene family may explain in part the previously reported changes in HMGR enzyme activities following wounding and elicitor treatment. The suppression of hmg1 and the enhancement of hmg2 and hmg3 transcript levels following elicitor treatment or inoculation with the incompatible race parallel the suppression in steroid and stimulation of sesquiterpenoid accumulations observed in earlier investigations. The results are discussed in relation to the hypothesis that there are discrete organizational channels for sterol and sesquiterpene biosynthesis in potato and other Solanaceous species.

Regulation of Em Gene Expression in Rice : Interaction between Osmotic Stress and Abscisic Acid.

Expression of the Em gene was characterized in rice (Oryza sativa L.) suspension cultures following exposure of the cultures to various combinations of abscisic acid (ABA) and salt. Response-saturating concentrations of either ABA (50 micromolar) or NaCl (0.4 molar) rapidly induced (by 60 minutes) the accumulation of Em mRNA, with a maximum accumulation occurring 12 to 24 hours after treatment. NaCl-induced Em expression was accompanied by a doubling of endogenous ABA levels as determined by immunoassay. Inhibition of ABA biosynthesis by fluridone during NaCl treatment reduced the levels of endogenous ABA by fourfold and Em expression by 50%. Desiccation of the cultures to 12 to 15% of their initial fresh weight increased endogenous ABA more than twofold and was accompanied by an increase in Em mRNA levels. Exposure of the cultures to heat shock temperatures, chilling, or ultraviolet light neither increased endogenous ABA levels nor induced Em expression. When a subthreshold or saturating level of NaCl was added in combination with increasing levels of ABA, Em transcripts were detected at ABA concentrations that alone did not induce expression of Em. Treatment with saturating levels of both NaCl and ABA resulted in a doubling of Em transcript levels over the maximum signal for each treatment alone. Hence, our data suggested that salt interacted synergistically with ABA, in part because of the increased sensitivity of rice cells to ABA. The effect of salt stress on Em gene expression in rice suspension cells appeared to operate through two pathways: one is mediated through increases in the level of ABA; the other is via a unique salt response pathway that includes an intermediate that is common to both the salt and ABA response chains.

Rapid in situ assay for indoleacetic Acid production by bacteria immobilized on a nitrocellulose membrane.

We have developed a new assay that differentiates between indoleacetic acid (IAA)-producing and -nonproducing bacteria on a colony plate lift. Medium supplemented with 5 mM L-tryptophan is inoculated with isolates of interest, overlaid with a nitrocellulose membrane, and then incubated until bacterial colonies reach 1 to 2 mm in diameter. The membrane is removed to a filter paper saturated with Salkowski reagent and incubated until distinct red haloes form around the colonies. The colorimetric reaction to IAA is limited to a region immediately surrounding each colony, is specific to isolates producing IAA, occurs within 1 h after the membrane is placed in the reagent, and is sensitive to as little as 50 pmol of IAA in a 2-mm spot. We have used this assay for quantifying epiphytic and endophytic populations of IAA-producing isolates of Pseudomonas syringae subsp. savastanoi and for detecting IAA-producing colonies of other pseudomonads and Erwinia herbicola. The assay provides a rapid and convenient method to screen large numbers of bacteria.

Involvement of 3-hydroxy-3-methylglutaryl coenzyme a reductase in the regulation of sesquiterpenoid phytoalexin synthesis in potato.

The importance of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in the regulation of sesquiterpenoid phytoalexin accumulation in potato (Solanum tuberosum L. cv Kennebec) was examined. Wounding of potato tubers produced a large temporary increase in HMG-CoA reductase activity of the microsomal and organelle fractions. Treatment of wounded tuber tissue with the sesquiterpenoid phytoalexin elicitor arachidonic acid further increased and prolonged the HMG-CoA reductase activity in the microsomal but not the organelle fraction. Incubation of elicitor-treated tuber tissue in white light reduced organelle and microsomal HMG-CoA reductase activity to 50% and 10%, respectively, of the activity of tissues held in darkness. Constant light also reduced overall phytoalexin accumulation 58% by greatly reducing levels of lubimin. Rishitin accumulation was not significantly altered by light. Application of nanomolar amounts of mevinolin, a highly specific inhibitor of HMG-CoA reductase, to elicitor-treated tuber tissue produced a large decline in lubimin accumulation and did not markedly alter rishitin accumulation. These results indicate that HMG-CoA reductase has a role in the complex regulation of sesquiterpenoid phytoalexin accumulation in potato.

Factors affecting the elicitation of sesquiterpenoid phytoalexin accumulation by eicosapentaenoic and arachidonic acids in potato.

Eicosapentaenoic and arachidonic acids in extracts of Phytophthora infestans mycelium were identified as the most active elicitors of sesquiterpenoid phytoalexin accumulation in potato tuber slices. These fatty acids were found free or esterified in all fractions with elicitor activity including cell wall preparations. Yeast lipase released a major portion of eicosapentaenoic and arachidonic acids from lyophilized mycelium. Concentration response curves comparing the elicitor activity of the polyunsaturated fatty acids to a cell-free sonicate of P. infestans mycelium indicated that the elicitor activity of the sonicated mycelium exceeded that which would be obtained by the amount of eicosapentaenoic and arachidonic acids (free and esterified) present in the mycelium. Upon acid hydrolysis of lyophilized mycelium, elicitor activity was obtained only from the fatty acid fraction. However, the fatty acids accounted for only 21% of the activity of the unhydrolyzed mycelium and the residue did not enhance their activity. Centrifugation of the hydrolysate, obtained from lyophilized mycelium treated with 2n NaOH, 1 molarity NaBH(4) at 100 degrees C, yielded a supernatant fraction with little or no elicitor activity. Addition of this material to the fatty acids restored the activity to that which was present in the unhydrolyzed mycelium. The results indicate that the elicitor activity of the unsaturated fatty acids is enhanced by heat and base-stable factors in the mycelium.

Eicosapentaenoic and Arachidonic Acids from Phytophthora infestans Elicit Fungitoxic Sesquiterpenes in the Potato.

Mycelial extracts from Phytophthora infestans caused necrosis and elicited the accumulation of antimicrobial stress metabolites in potato tubers. A portion of the material with elicitor activity could be extracted from the mycelium by a mixture of chloroform and methanol. The most active elicitors of stress metabolites in these extracts were eicosapentaenoic and arachidonic acids. These fatty acids were found in either free or esterified form in all active fractions of the mycelial extracts.