Monoterpene and Phenolic Compound Concentrations in Water-Stressed Red Pine Inoculated with Sphaeropsis sapinea

ABSTRACT Changes in monoterpene and phenolic compounds resulting from water stress and colonization by Sphaeropsis sapinea were examined for 9- and 11-year-old red pine trees in a plantation and 3-year-old seedlings in a growth chamber. Four treatments were assigned at random to individual trees in the field: no treatment, herbicide to kill surrounding weeds, supplemental water, and both herbicide and supplemental water. In the growth chamber, seedlings were either not watered (water stressed) or watered daily (nonstressed). Shoots were inoculated with agar plugs colonized with either S. sapinea isolates of morphotype A and B (field) or only isolates of morphotype A (growth chamber). Nine monoterpenes were detected in tissue extracts; the most common were alpha-pinene (59 to 74% of the total), beta-pinene (13 to 33% of the total), and delta-3-carene (1 to 5% of the total). Shoots inoculated with isolates of morphotype A had more severe symptoms and produced higher concentrations of monoterpenes in both experiments compared with the controls. In the growth chamber, inoculations with isolates of morphotype A caused higher concentrations of phenolics compared with the controls. In the field experiment, monoterpenes increased in quantity only in shoots of stressed trees inoculated with isolates of morphotype A. Isolates of morphotype B caused few symptoms and did not alter monoterpene concentrations. Increases in monoterpenes do not appear to be involved in the response to infection by morphotype A in nonstressed trees, and the role of phenolics is unclear. However, these results are consistent with previous observations that monoterpenes may be involved in the differences in aggressiveness between morphotypes on red pine.     

Sphaeropsis sapinea and Water Stress in a Red Pine Plantation in Central Wisconsin

ABSTRACT A study was conducted to determine the effects of water stress resulting from competing vegetation on disease development of Sphaeropsis sapinea in red pine plantations. A 9-year-old plantation was selected in 1992 and experiments were conducted for three consecutive years. Four treatments were assigned at random to individual trees: no treatment, herbicide to kill surrounding weeds, supplemental water, and both herbicide and supplemental water. Two isolates of each S. sapinea morphotype (A and B) were used to inoculate wounded lateral shoots. Disease development was measured as the maximum distance below the inoculation site at which necrotic needles were observed. Nonwatered trees with competing vegetation (nontreated condition) had significantly lower predawn needle water potentials (more water stress) and more severe disease development than trees that received the herbicide, water, or combined herbicide and water treatments. The most severe disease occurred in the driest year and the least in the wettest year. Competing vegetation indirectly affected disease development by inducing water stress, even in relatively moist years, on trees previously considered well established. Isolates of morphotype A were more aggressive than isolates of morphotype B. Conclusions from this research have implications for sustainable management of the region's conifer forests.     

Effects of Moderate Water Stress on Disease Development by Sphaeropsis sapinea on Red Pine

ABSTRACT The aggressiveness of Sphaeropsis sapinea isolates was compared on water-stressed and nonstressed 3-year-old red pines (Pinus resinosa) in greenhouse and growth chamber experiments. Water was withheld from stressed seedlings to achieve mean predawn needle water potentials (psi(PD)) above -1.9 MPa. The lowest mean psi(PD) of well-watered seedlings was maintained at or above -0.8 MPa. Young shoots were inoculated by placing colonized agar plugs on wounds made by removing a needle fascicle. Two isolates of each recognized morphotype (A and B) were used in the greenhouse experiment and two isolates of morphotype A were used in the growth chamber experiment. After 4 weeks, isolates of morphotype A caused more severe symptoms and could be recovered farther from the inoculation site on water-stressed than on nonstressed trees in both experiments. In the greenhouse experiment, isolates of mor-photype A also caused more severe symptoms and could be recovered farther from the inoculation site than isolates of morphotype B, regardless of watering regime. These results indicate that water stress at levels observed typically in the field can result in increased disease development by isolates of S. sapinea morphotype A on red pine. The reduction of water stress of red pines in the field may reduce losses due to Sphaeropsis shoot blight.     

Detection of Latent Sphaeropsis sapinea Infections in Austrian Pine Tissues Using Nested-Polymerase Chain Reaction

ABSTRACT Sphaeropsis sapinea is the causal agent of Sphaeropsis tip blight disease of pines. Past surveys of diseased and symptomless Austrian and Scots pines revealed that latent infections of symptomless shoots by S. sapinea are common. The role of these latent infections in the tip blight disease is unknown. A sampling technique and nested-polymerase chain reaction (PCR) protocol were developed to detect latent S. sapinea in symptomless pine shoots. The sampling protocol was designed to be minimally destructive to the shoot so it could be preserved for further studies. The primers that were developed were specific for S. sapinea DNA and did not amplify DNA from any of 13 other endophytic fungal species that were commonly isolated from symptomless pine shoots. The PCR primers also amplified DNA of Botryosphaeria obtusa, which was, however, rare in symptomless Austrian pine tissues. The protocol detected as little as 0.93 pg of S. sapinea DNA in terminal bud samples and 10.4 pg of DNA in bark samples. Correlation (chi-square) analyses indicated that the nested-PCR protocol detected latent S. sapinea infections in both bud and bark samples with an efficiency that was statistically equivalent to isolating the fungus from the tissue. The nested-PCR protocol will make it possible to more quickly identify latent S. sapinea infections in symptomless pine shoots and should be useful in future studies of the latency phenomenon.     

Occurrence, isolation and biological activity of phytotoxic metabolites produced in vitro by Sphaeropsis sapinea, pathogenic fungus of Pinus radiata

Sphaeropsis sapinea was repeatedly isolated in Sardinia from symptomatic samples of the upper part of declining pine (Pinus radiata) plants. Observed symptoms mainly consisted of foliage chlorosis, drying of needles and cankers on branches. The S. sapinea strains were shown to produce phytotoxic metabolites in culture filtrates. Three metabolites were isolated for the first time from this fungus and identified by their spectroscopic and optical properties as R-(−)-mellein, (3R,4R)-4-hydroxymellein and (3R,4S)-4-hydroxymellein. When assayed for phytotoxic and antifungal activities on host and non-host plants and on some phytopathogenic fungi, the R-(−)-mellein showed significant activity, while the other two 3,4-dihydroisocoumarins showed only a synergic activity in both tests.     

Effects of fertilization with ammonium sulphate and potassium sulphate on the development of Sphaeropsis sapinea in Corsican pine

Potted plants ofPinus nigra subsp.laricio were fertilized with ammonium sulphate (AS) and potassium sulphate (PS) each year during 3 consecutive years. The then 5-year-old plants were artificially inoculated withSphaeropsis sapinea. The fungus caused much bigger bark necroses in plants that had been fertilized with AS, than in plants fertilized with PS. It is concluded thatP. nigra growing in poor sandy soil becomes more susceptible toS. sapinea when fertilized with an excess of AS. This may explain partly the increased incidence ofS. sapinea inP. nigra stands in areas with a high rate of atmospheric NH₄-deposition in the Netherlands.     

Bioassays on Leptographium wingfieldii, a Bark Beetle Associated Fungus, with Phenolic Compounds of Scots Pine Phloem

The fungicidal activity of five phenolic metabolites occurring in Scots pine phloem (Cat, Tax, PS, PC and PSM) and of phenolic extracts from unwounded (fresh) and wounded phloem of Scots pine were measured against Leptographium wingfieldii, a fungus associated with Tomicus piniperda. Bioassays were performed in micro-wells in a standard medium in the absence and presence of Scots pine phloem. In both media, methanol extracts from the unwounded and wounded phloem, PS, PSM and PC inhibited fungal growth. Tax and Cat had no effect or stimulated fungal growth. The presence of Scots pine phloem in the liquid medium reduced the inhibitory effect of phenolic compounds. At low concentrations (10–4–10–5M), the fungus seemed to be able to degrade PS, PSM and PC while, whatever the Cat and Tax concentrations, it did not seem to degrade them. These results suggest that three phenolic compounds have a potential fungitoxicity in vitro and that, in vivo, these phenols could play a role in the efficiency of the induced reaction to stop fungal growth.     

洋葱伯克氏菌群不同基因型菌株对几种重要植物病原真菌的抑制作用及其潜在致病性

从玉米和水稻根围分离到70株不同基因型的洋葱伯克氏菌,对这些菌株进行了拮抗植物病原菌的筛选,并对高拮抗菌株进行了潜在致病性和安全性分析。结果表明,有46株洋葱伯克氏菌对一种或多种病菌有较高的拮抗活性。在不同的洋葱伯克氏菌基因型内,以基因型Ⅴ内拮抗菌株占的比例最高,对所测5种病原真菌的平均拮抗菌株比率为80．0％,其中部分菌株表现出很强的拮抗活性。筛选出的4株高拮抗菌株对洋葱不具有致病性,同时也未检测到与人体致病相关的BCESM毒力基因。     

Inhibition of acetyl-coenzyme a carboxylase by coenzyme a conjugates of grass-selective herbicides

A total of 146 samples of different kinds of cheeses produced in Spain were analysed in order to ascertain the specific contamination pattern. The organochlorine compounds studied were those most commonly investigated in previous surveys: α-HCH, β-HCH, γ-HCH (lindane), γ-HCH, chlordane, aldrin, dieldrin, endrin, heptachlor, heptachlor epoxide, and the isomers and metabolites of DDT. α-HCH, β-HCH, γ-HCH, chlordane, p,p′, DDT, and p,p′-DDE were found in more than 76% of samples; p,p′-DDE and γ-HCH were the most frequently detected, with frequencies of 100 and 97.9% respectively. γ-HCH, aldrin, dieldrin, heptachlor, heptachlor epoxide, o,p′-DDT, p,p′-DDD and o,p′-DDD were observed at lower frequencies. No residues of endrin were detected in any sample. Insecticides exceeding the maximum residue limits (MRLs) were chlordane, β-HCH, α-HCH and γ-HCH, with 42, 20, eight and six samples respectively. Mean residues of organochlorines found were as follows (μ kg^?1 butterfat): α-HCH = 46.3; β-HCH = 46.5; γ-HCH = 54.2; δ-HCH = 16.9; aldrin = 16.7; dieldrin = 9.7; heptachlor = 15.9; heptachlor epoxide = 14.8; chlordane = 50.2; o,p′-DDT = 5.1; p,p′-DDT = 12.4; o,p′-DDT = 19.6; p,p′-DDD = 46.7; o,p′-DDE = 6.9; p,p′-DDE = 40.7 (.DDT = 55.0). Estimated dietary intakes (EDIs) from cheese consumption were compared to acceptable daily intakes (ADIs) for the pesticides where residues exceeded the MRL. EDIs calculated were in all cases below ADIs, and, therefore, based on the ADIs, there is no health risk involved in the consumption of cheese from Spain arising from organochlorine residues.     

Differential accumulation of fenarimol by a wild-type isolate and fenarimol-resistant isolates of Penicillium italicum

Accumulation of [¹⁴C] fenarimol by mycelium ofPenicillium italicum was studied with isolates having varying levels of laboratory resistance to fenarimol. All resistant isolates tested showed a significantly lower accumulation than the wild-type isolate.Various metabolic inhibitors enhanced accumulation to relatively high levels in both wildtype and resistant isolates. it indicates that accumulation is governed by two processes viz. a non-mediated influx and an energy-dependent efflux. A relatively high fenarimol efflux in resistant isolates probably accounts for low accumulation and for fenarimol resistance. One of the inhibitors which annihilated fenarimol efflux and enhanced fenarimol accumulation was sodium orthovanadate. The synergistic action of fenarimol and orthovanadate to both wildtype and resistant isolates in crossed-paper strip bioassays is probably related to the effect of the latter compound on fenarimol accumulation. Synergistic action between the chemicals in control ofPenicillium decay of oranges could not be detected.Samenvatting De accumulatie van [¹⁴C]fenarimol door mycelium vanPenicillium italicum werd bestudeerd bij isolaten met een uiteenlopende graad van laboratorium-resistentie tegen fenarimol. Alle getoetste resistente isolaten vertoonden een lagere opname dan de wild-stam.Verschillende antimetabolieten verhoogden de accumulatie tot relatief hoge waarden die voor gevoelige en resistente isolaten ongeveer gelijk waren. Deze waarneming duidt erop dat accumulatie wordt bepaald door twee processen: nonmediated influx en energie-afhankelijke efflux. Een hogere fenarimol efflux in resistente isolaten vormt waarschijnlijk de verklaring voor de lagere accumulatie en voor het resistentiemechanisme. Een van de remmers die de fenarimolefflux te niet doet, en de accumulatie van fenarimol verhoogt, is natriumorthovanadaat. De synergistische werking van fenarimol en orthovanadaat tegen zowel wild-type als resistente isolaten in crossed-paper strip biotoetsen houdt waarschijnlijk verband met het effect van laatstgenoemde stof op de accumulatie van fenarimol. Synergistische werking van deze verbindingen bij de bestrijding vanPenicillium-rot op sinaasappels werd niet waargenomen.     

Differential regulation of MBP kinases by a glycoproptein elicitor and a polypeptide suppressor from Mycosphaerella pinodes in pea

A polypeptide fungal suppressor from a pea pathogen Mycosphaerella pinodes plays a key role in pathogenesis by suppressing elicitor-induced defense response(s) in pea (Pisum sativum L). In this study, we show that treatment of pea tissues with the polysaccharide elicitor secreted by M. pinodes results in rapid increased activation of two myelin basic protein (MBP)-dependent kinases p44 (≈44 kDa) and p48 (≈48 kDa) within 15–30 min upon elicitation. Interestingly, the suppressor inhibited the elicitor-induced activation of only p44 kinase. While the defense-inducing signalling molecules, chitosan and salicylic acid (SA) activated the p44 and p48 kinases, methyl jasmonate (MeJA) did not. The abiotic stress signals, abscisic acid (ABA), NaCl and wounding activated the p48 kinase alone. These results demonstrate that MAPKs are differentially activated in response to pathogen invasion and abiotic stress in pea. Furthermore, specific inhibition of elicitor-induced p44 kinase activation by a MAPKK inhibitor, PD098059 and protein kinase inhibitor, K252a correlated with the suppression of elicitor-induced phenylalanine ammonia lyase (PAL) gene expression, supporting a role for p44 in the elicitor-induced defense response(s) in pea. Inhibition of p44 by the phosphoinositide (PI) turnover inhibitor, neomycin (a fungal suppressor mimic), and potentiation of p44 by the diacylglycerol (DAG) kinase inhibitor, R59022 indicated that p44 may be acting downstream of (PI) metabolism. Taken together, our results indicate that suppressor of defense elicitation from M. pinodes acts through inhibition of a MAPK (p44), possibly through a PI signaling pathway, facilitating the establishment of basic compatibility during infection of pea.     

Inhibition of mycelial growth and sporidial formation in the wheat pathogenNeovossia indica by a polyamine biosynthetic inhibitor

DL-α-Difluoromethylornithine (DFMO), a specific suicide inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase (EC 4.1.1.17), strongly inhibited mycelial growth and sporidial formation of the wheat pathogen,Neovossia indica, in vitro, while DL-α-difluoromethylarginine (DFMA), the analogous suicide inhibitor of arginine decarboxylase (EC 4.1.1.19), did not. The inhibited mycelial growth and sporidial formation were not only restored by putrescine (polyamine) addition, but were actually enhanced in the putrescine + DFMO cultures. Besides altering mycelial growth and morphology, DFMO also reduced the cell size drastically. The inhibition of fungal polyamine biosynthesis is discussed in relation to selective control of plant disease.     

Inhibition of aflatoxin production in Aspergillus flavus by pyrazolines and pyrazoles

A new series of 1,3,5-triarylpyrazolines, obtained from 3, 4-dichloroacetophenone, were readily dehydrogenated by tetrachloro-o-benzoquinone at room temperature to the pyrazoles. The spectra and fluorescence of the compounds are discussed. The pyrazolines and pyrazoles did not significantly affect the mycelial growth of 19 fungal isolates, but at low concentrations, they inhibited aflatoxin production by Aspergillus flavus.     

Defense Mechanism of Rice Plant to Respiratory Inhibition by a Promising Candidate Blasticide, SSF126

A new candidate systemic rice blasticide, SSF126, dose-dependently inhibited NADH oxidation by submitochondrial particles from rice roots. However, oxidation by the root submitochondrial particles was much less susceptible to SSF126 compared to that by submitochondrial particles from mycelial cells ofPyricularia grisea, a pathogen causing rice blast. Interestingly, SSF126 did not completely suppress the respiration by intact rice roots, and the respiratory activity of the roots recovered from inhibition time-dependently even in the presence of SSF126 at concentrations sufficient to fully block oxidation by the submitochondrial particles. This recovery was not due to selective extrusion of SSF126 from the roots, but to switching from the cytochrome pathway to the alternative cyanide-resistant respiratory pathway. In immunoblots of the alternative oxidase, high molecular mass species were detected in the mitochondria from rice roots in addition to low molecular mass species. Quantification of high and low molecular mass species revealed an increase in the amount of a protein corresponding to a 36-kDa species equivalent to a decrease in the amount of a protein corresponding to a 72-kDa species following a 5-h incubation with SSF126. This conversion of the alternative oxidase to the low molecular mass species in the mitochondria was correlated with the respiratory recovery found in intact rice roots, suggesting that the low molecular mass species is the active form of the alternative oxidase and the high molecular mass species is the inactive form. These results suggest that rice plants can block the severe injury caused by limiting the cytochrome pathway by SSF126 through utilization of the alternative pathway promoted by the interconversion of the alternative oxidase protein.     

Translation Inhibition of Capped and Uncapped Viral RNAs Mediated by Ribosome-Inactivating Proteins

ABSTRACT Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove specific purine residues from the sarcin/ricin (S/R) loop of the large rRNA and arrest protein synthesis at the translocation step. In addition to their enzymatic activity, RIPs have been reputed to be potent antiviral agents against many plant, animal, and human viruses. We recently showed that pokeweed antiviral protein (PAP), an RIP from pokeweed, inhibits translation in cell extracts by binding to the cap structure of eukaryotic mRNA and viral RNAs and depurinating these RNAs at multiple sites downstream of the cap structure. In this study, we examined the activity of three different RIPs against capped and uncapped viral RNAs. PAP, Mirabilis expansa RIP (ME1), and the Saponaria officinalis RIP (saporin) depurinated the capped Tobacco mosaic virus and Brome mosaic virus RNAs, but did not depurinate the uncapped luciferase RNA, indicating that other type I RIPs besides PAP can distinguish between capped and uncapped RNAs. We did not detect depurination of Alfalfa mosaic virus (AMV) RNAs at multiple sites by PAP or ME1. Because AMV RNAs are capped, these results indicate that recognition of the cap structure alone is not sufficient for depurination of the RNA at multiple sites throughout its sequence. Furthermore, PAP did not cause detectable depurination of uncapped RNAs from Tomato bushy stunt virus (TBSV), Satellite panicum mosaic virus (SPMV), and uncapped RNA containing poliovirus internal ribosome entry site (IRES). However, in vitro translation experiments showed that PAP inhibited translation of AMV, TBSV, SPMV RNAs, and poliovirus IRES dependent translation. These results demonstrate that PAP does not depurinate every capped RNA and that PAP can inhibit translation of uncapped viral RNAs in vitro without causing detectable depurination at multiple sites. Thus, the cap structure is not the only determinant for inhibition of translation by PAP.     

Inhibition of insect pest α-amylases by little and finger millet inhibitors

The inhibitory effect of three proteinaceous inhibitors isolated from little and finger millet was examined on gut α-amylases for four stored grain and four phytophagous insect-pests. Additionally, using native PAGE, several α-amylases isozymes were observed in all insect-pests studied. Furthermore, thermostabilities and the pH optimum for insect-pests α-amylases, which varied from acidic to alkaline, were also determined. On the other hand, proteinaceous inhibitors from little millet seeds Panicum sumatrense (LMCO3) and from finger millet (FMCO11 and FMCO13) inhibited insect-pests α-amylases with different proportions. The highest inhibition percent was recorded for LMCO3 and FMCO13 against Callosobruchus chinensis α-amylase, where the inhibition percent was approximately 70 and 50%, respectively. Furthermore, millet α-amylase inhibitors also reduced significantly digestive α-amylolytic activities of Acaea janata, C. cephalonica, Sitophilus oryzae, and Tribolium castaneum, indicating that these α-amylase inhibitors could be used toward crop insect-pests.     

Inhibition by aluminum of mycelial growth and of sporangial production and germination inPhytophthora infestans

Mycelial growth on clarified V8 agar of the potato late blight pathogenPhytophthora infestans was inhibited when either aluminum chloride (AlCl₃, 6 H₂O) or aluminum sulfate (Al₂(SO₄)₃, 18 H₂O) was added to the culture medium at concentrations of 2.5–100 mg.l^–1 Al³⁺. Toxicity of Al³⁺ varied among the fiveP. infestans isolates tested, but toxicity of sulfate and chloride salts was similar for a given isolate. Overall sporangial production was affected in all five isolates by both Al³⁺ forms. Al³⁺ also decreased sporangial germination at concentrations equal to or greater than 10 mg.l^–1 in two isolates. These data support the hypothesis of aluminum toxicity as a major factor in soil suppressiveness toP. infestans.