Leaf Disc Assay Research Articles

A Leaf Disk Assay for Screening Sweet Cherry Genotypes for Susceptibility to Powdery Mildew

A detached leaf disk assay for screening sweet cherry (Prunus avium L.) genotypes for susceptibility to powdery mildew (PM) [Podosphaera clandestina (Wallr.:Fr.) Lev.] was developed by evaluating the effects of photoperiod (24 hours light, 0 hours light, 14 hours light/10 hours dark), substrate nutrient content (sterile distilled water, 1% sucrose), leaf age (old, young, emergent), and leaf explant size (intact leaf, 30 mm, 20 mm) on PM growth on leaves from the susceptible cultivar Bing. The only parameter described that had a significant (P ≤ 0.001) effect on PM growth was leaf age. Old leaves, designated as the third fully expanded leaf from the basal end of current-year's shoot growth, were never infected with PM under controlled inoculations. In the absence of significant differences between treatments, those parameters with the highest treatment means were selected for subsequent evaluation. To test the leaf disk assay, 14 sweet cherry cultivars were screened in two experiments, and rated according to level of PM susceptibility. Rank sum comparison of results from cultivars used for leaf disk screening agreed with earlier field rankings of the same cultivars. The developed leaf disk assay greatly reduced the space required to screen sweet cherry cultivars, and was a repeatable and objective predictor of field resistance that may be useful for screening germplasm or breeding populations.

Distribution of Baseline Sensitivities to Azoxystrobin Among Isolates of Plasmopara viticola.

An excised leaf disc assay was developed for determining the sensitivity of isolates of Plasmopara viticola (causal agent of grapevine downy mildew) to the strobilurin fungicide azoxystrobin. Five repeated assays with the same five single-sporangiophore isolates showed that the technique yielded reproducible results; that is, coefficients of variation ranged from 4.0 to 20% (mean 12%) for effective doses for 50% control (ED50 values) based on disease incidence and from 4.4 to 14% (mean 8.1%) for ED50 values based on disease severity. Then, the assay was applied to 81 single-sporangiophore isolates of P. viticola collected from 10 geographically distinct vineyards in western New York, providing a baseline distribution of sensitivities within this population. For disease incidence, individual ED50 values ranged from 0.05 to 0.94 μg/ml (mean 0.40 μg/ml), whereas for disease severity they ranged from 0.04 to 0.78 μg/ml (mean 0.24 μg/ml). When 61 of these isolates were similarly tested at a single discriminatory dose of 0.50 μg/ml, azoxystrobin provided 61% control of disease incidence versus 41 and 1.1% control for trifloxystrobin and kresoximmethyl, respectively; for disease severity (colony diameter), azoxystrobin provided 80% control versus 57 and 1.1% control for trifloxystrobin and kresoximmethyl, respectively. These results provide information that can be utilized in future monitoring of P. viticola resistance to azoxystrobin and indicate differences in the intrinsic activities of the three strobilurin fungicides against this pathogen.

FIELD BIOASSAY METHODOLOGIES FOR THE DETECTION OF RESISTANCE TO ACARICIDES IN POPULATIONS OF EUROPEAN RED MITE (ACARI: TETRANYCHIDAE)

AbstractThe toxicity of dicofol, pyridaben, formetanate HCl, and abamectin to European red mite, Panonychus ulmi (Koch), was determined using either a Petri dish or leaf dip bioassay (as potential field assay procedures) and was compared with results from a standard laboratory procedure that requires a Potter spray tower to treat leaf disks. Abamectin and pyridaben were the most toxic acaricides tested, and dicofol and pyridaben were more toxic when applied to Petri dishes than to leaf disks. Formetanate HCl was the least toxic acaricide tested by either leaf disk or leaf dip procedures. Diagnostic concentrations of pyridaben were determined as 5 mg active ingredient per litre (a.i./L) with the Petri dish assay and 15 mg a.i./L with the leaf disk assay. Mortality of European red mites to pyridaben at diagnostic concentrations was consistently 100% with the leaf disk assay and > 90% with the Petri dish assay. Residues of pyridaben on Petri dishes stored at 4 °C in a refrigerator were stable for at least 21 d. For dicofol, both leaf disk and Petri dish assays gave similar results, and 25 mg a.i./L, previously used as the diagnostic concentration in the Petri dish assay, resulted in > 99% mortality of the standard susceptible strain. This technique identified resistant individuals in all of the field populations tested. Abamectin applied at 2 mg a.i./L to leaf disks consistently gave 98–100% mortality, but results at 2 mg a.i./L with the Petri dish assay gave variable results and this was not a reliable technique for monitoring resistance. Concentrations of formetanate HCl required for the leaf dip assay were too high to be useful in field assays; however, all populations of European red mite tested using the leaf disk assay appeared susceptible. For the ovicide, clofentezine, mortality of eggs tested by the leaf disk assay at 100 or 500 mg a.i./L ranged from 89 to 100%. Results with the leaf dip procedure, using a 350 mg a.i./L suspension consistently resulted in > 96% mortality of eggs from susceptible field populations. These procedures are a useful tool in the development of resistance management programs for acaricides used in integrated pest management (IPM) systems for tree fruits.

Differences in feeding response among three cucurbitaceous feeding leaf beetles to cucurbitacins.

Feeding responses to four cucurbitacins (B, E, I and E-glucoside) were investigated in three cucurbitaceous feeding leaf beetles, Aulacophora femoralis (Motschulsky), A. lewisii Baly and A. nigripennis Motschulsky by filter paper assay. A. femoralis and A. lewisii were strongly stimulated to feed by four cucurbitacins, and A. nigripennis was weakly stimulated to feed by cucurbitacin B at a concentration of only 0.01 mg/ml. A. nigripennis showed no significant feeding stimulant activity to cucurbitacins by a leaf disk assay. These results indicate that not all cucurbitaceous feeding beetle species are strongly stimulated to feed by cucurbitacins.

Geographic Variation of White Pine Blister Rust Aeciospore Infection Efficiency and Incubation Period

Frequency of infection, main effects and interactions among four geographic sources of white pine blister rust (WPBR) (Cronartium ribicola J.C. Fisch.) and 10 Ribes-sites (Ribes L. specie × site combinations) was investigated using leaf disk assay. Two clones, one of R. hudsonianum (Dougl.) Jancz. and one of R. viscosissimum Prush., were not infected by any sources of WPBR. One clone of R. viscosissimum that was not infected by two sources of WPBR was susceptible to the other sources. Highly significant WPBR sourc × Ribes-site analysis of variance interaction for incubation period and infection efficiency precluded testing main effects. Profile plots of incubation period interaction means showed orderly interaction by all WPBR sources and plots of infection efficiency showed that aggressively virulent WPBR from Oregon (Champion Mine) ranked near or at the bottom for infection efficiency for all Ribes-sites. Meanwhile, aggressively virulent WPBR from Idaho (Merry Creek) ranked near the bottom for infection efficiency when inoculated onto Ribes obtained from the Cascade Mountains but switched to the highest ranking when inoculated onto Ribes obtained from Idaho. Geographic interaction of white pine blister rust and Ribes for incubation period and infection efficiency may help to explain geographic patterns recently observed in WPBR molecular markers.

Two Field Isolates of Tomato Spotted Wilt Tospovirus Overcome the Hypersensitive Response of a Pepper (Capsicum annuum) Hybrid with Resistance Introgressed from C. chinense PI152225.

The hypersensitive response to tomato spotted wilt tospovirus (TSWV) present in Capsicum chinense PI152225 (1) was introgressed into C. annuum cultivars. During the summer of 1998, a hybrid with good agronomic performance was grown in glasshouses in Albenga, Liguzia Region of northwestern Italy, an area where infection by TSWV in pepper has been severe since 1992. In August, observations of different susceptible cultivars revealed that >50% of plants had TSWV-like symptoms, whereas the resistant hybrid remained healthy, except for two plants that showed virus-like symptoms on apical leaves and fruits. From the infected plants, tospoviruses (coded P164/6 and P166) were transmitted by sap-inoculation to Nicotiana benthamiana. Triple-antibody sandwich enzyme-linked immunosorbent assay with a panel of monoclonal antibodies against the TSWV nucleocapsid, but with different reactivity to the related species groundnut ringspot (GRSV) and tomato chlorotic spot (TCSV) viruses, indicated the isolates were TSWV. The host ranges of the isolates were wide and typical of normal TSWV isolates. Thus, they incited typical symptoms in all 50 TSWV-susceptible C. annuum cv. Quadrato d'Asti plants. However, isolate P164/6 also systemically infected 12 of 27 C. chinense PI152225 and 14 of 19 C. chinense PI159236 plants. These accessions are normally resistant to TSWV (1). Isolate P166 systemically infected 7 of 17 C. chinense PI152225 and 6 of 11 C. chinense PI159236 plants. Systemically infected plants showed severe necrosis, and some plants died. Other plants showed only necrotic local lesions. The response by C. chinense differed from that caused by typical TSWV, which causes only local lesions, and from both GRSV and TCSV, which cause mosaic but no necrosis in 100% of plants. The two new TSWV isolates were tested for transmission using a local population of Frankliniella occidentalis in a leaf disk assay with susceptible C. annuum. Transmission rates were high: 93.7% (63 thrips) for isolate P164/6 and 89.9% (49 thrips) for P166. Thus, the fitness of the two TSWV resistance-breaking isolates (a wide experimental host range and high transmission rates by the natural vector) was as high as that of typical TSWV. The absence of systemic infection in some C. chinense PI152225 and PI159236 plants that are resistant to typical TSWV suggests the possibility of selecting plants resistant to these pathotypes. This is the first report of field tospovirus isolates typed as TSWV (according to the current taxonomy based on nucleocapsid serology) overcoming the hypersensitive response of C. chinense PI152225 and PI159236, an ability previously found only in closely related viruses: TCSV and GRSV (2). Other TSWV-like isolates systemic on C. chinense were not typed further (3,4).

The Identification of the Vector Species of Iris Yellow Spot Tospovirus Occurring on Onion in Brazil.

In Brazil, tospoviruses have been reported in several horticultural and ornamental plants. In the northeast region of Brazil, a tospovirus has emerged as a devastating virus on onion cultures. Based on serology and the sequence of nucleocapsid (N) protein gene, this pathogen was identified as a strain of iris yellow spot tospovirus (IYSV) (1). This virus was first identified on iris and leek in The Netherlands and later on onion in Israel. For an effective integrated management of tospoviruses in Brazil, identification of IYSV vector is essential. Three thrips species, Thrips tabaci, Frankliniella schultzei, and F. occidentalis, that are major vegetable and floral crop pests in the Federal District, Brazil, were tested for their ability to transmit the virus by leaf disk assay (2). All thrips, up to 8 h old, were given an acquisition access period of 48 h at 25°C on IYSV-infected Nicotiana benthamiana plants in Tashiro-cages. Thrips were then reared on uninfected Datura stramonium detached leaves until the adult stage. These adults were transferred individually to microcentrifuge tubes containing an N. benthamiana leaf disk and were incubated for 48 h for virus inoculation. The leaf disks were then incubated 4 more days to allow development of the virus infection, and the presence of virus was evaluated by Dot-enzyme-linked immunosorbent assay (Dot-ELISA) with polyclonal antibodies against N protein of IYSV. Adult thrips were also used for direct inoculation to N. benthamiana plants, three thrips per plant. By the leaf disk assay, 45.8% (22 out of 48) of T. tabaci transmitted the virus, but F. schultzei (n = 48) and F. occidentalis (n = 32) did not transmit it. All plants (4 out of 4) directly inoculated by T. tabaci showed symptoms and infection by Dot-ELISA, while no plants inoculated with F. schultzei (n = 5) and F. occidentalis (n = 3) were positive, either by symptom observation or by Dot-ELISA. Only T. tabaci showed potential for a high capacity to transmit the IYSV onion isolate. In the field, considering the host preference of thrips, T. tabaci was considered the most important vector species of IYSV on onion.

Comparison of Leaf Disk, Greenhouse, and Field Screening Procedures for Evaluation of Grape Seedlings for Downy Mildew Resistance

This research was conducted to compare an in vitro leaf disk technique with greenhouse and field evaluations for screening large populations of grape (Vitis sp.) seedlings for downy mildew (Plasmopara viticola Berk. & Curt. Berl. & de Toni) resistance. Seedlings produced by crossing resistant × resistant, resistant × susceptible, and susceptible × susceptible parents were rated for sporulation, chlorosis, and necrosis. Leaf disk sporulation ratings at the first and second rating were highly correlated with the second sporulation rating in the field. Necrosis ratings from the leaf disk evaluations were significantly correlated with field necrosis ratings, but leaf disk chlorosis ratings were not correlated with field ratings. Some correlations, including evaluations of chlorosis, between the greenhouse and field ratings were highly significant. Seedling ratings of 0 or 1 for sporulation, chlorosis, and necrosis in the leaf disk assay agreed with field evaluations 85.6% of the time vs. 80.3% agreement between greenhouse and field ratings. Sporulation was the parameter most highly correlated between leaf disk or greenhouse and field evaluation of resistance. The leaf disk procedure appeared to be a good predictor of field resistance, and is more practical than the greenhouse method for screening large populations.

Sensitivity of EuropeanPlasmopara viticolapopulations to cymoxanil

Between 1995 and 1997, 278 grape downy mildew (Plasmopara viticola) populations originating from European vineyards were characterised for their sensitivity to cymoxanil in a leaf-disc assay. The sensitivity profile revealed a wide distribution, with minimum inhibitory concentrations (MIC) ranging from 10 to more than 800 mg litre -1 . EC 50 values ranged from 1 to more than 800 mg litre -1 with an average of 125 mg litre -1 . The sensitivity distribution was stable between 1995 and 1997. Surprisingly, populations from Portugal appeared significantly more sensitive than those from France or Italy, which could not be linked to differential cymoxanil usage in these countries. P viticola populations collected outside Europe and never exposed to cymoxanil appeared significantly more sensitive than exposed European populations, with an average EC 50 value of 10 mg litre -1 . The level of sensitivity of European P viticola populations was relatively unaffected by the number of cymoxanil applications made during a season or by the number of years of cymoxanil use. No link was found between the level of sensitivity in the leaf-disc assay and the level of performance of the cymoxanil mixtures used in the fields where the populations originated. Specific field trials conducted in Italy and Portugal have shown that the performance of cymoxanil-based mixtures remained good even on populations of the grape downy mildew fungus characterised as less sensitive in the leaf-disc assay. While there are no baseline sensitivity data for pre-commercialisation P viticola populations, the results of our study suggest that a shift in sensitivity (12.5-fold) may have occurred in some areas since introduction of cymoxanil on grapes nearly 20 years ago. Because cymoxanil is never used alone, it is difficult to determine whether or not practical resistance is occurring in European vineyards.

Effect of Film-forming Polymers on Control of Lily Leaf Blight Caused by Botrytis elliptica

The effectiveness of film-forming polyelectrolytes for the control of lily leaf blight caused by Botrytis elliptica was evaluated using laboratory a leaf disk assay, greenhouse tests and field trials. Among the six polyelectrolytes, 400 ppm FO4240SH, FO4490SH and FO4550SH reduced the disease severity of lily leaf blight in leaf-disk tests. Both FO4240SH and FO4490SH also suppressed sporulation of the pathogen on leaf disks. In greenhouse tests, the number and size of lesions on leaves of Lilium oriental hybrid cv. Star Gazer were markedly reduced by FO4490SH and FO4550SH. Field trials showed that the effectiveness of FO4490SH was similar or better than that of procymidone on the reduction of lily leaf blight disease severity. The polymers had no harmful effects on the lily plants. The cationic polyelectrolytes FO4240SH, FO4490SH and FO4550SH reduced the percentage of conidial germination, inhibited germ-tube growth, and also suppressed the esterase production by germ tubes of B. elliptica. All the above evidence indicates that the disease control achieved with polyelectrolytes is due, at least in part, to the reduction of esterase secretion by B. elliptica.

A cDNA from tobacco codes for an inhibitor of virus replication (IVR)-like protein.

We have shown previously that localization of tobacco mosaic virus (TMV) in tobacco is associated with a ca. 23 kDa protein that inhibits replication of several plant viruses. This protein, named 'inhibitor of virus replication' (IVR), was purified from the medium of TMV-inoculated protoplasts derived from Nicotiana tabacum cv. Samsun NN. IVR was shown to be present also in induced-resistant leaf tissue of N. tabacum cv. Samsun NN. We prepared an expression cDNA library from such induced-resistant tissue and screened it with a polyclonal antibody raised against the IVR protein. A 1016 bp clone (named NC330) containing a 597 bp open reading frame, coding for a 21.6 kDa polypeptide, was isolated. The NC330 clone hybridized with RNA from induced-resistant tissue from N. tabacum cv. Samsun NN but not with RNA from non-induced tissue. Likewise, it did not hybridize with RNA from infected or uninfected tissue of N. tabacum cv. Samsun nn. Similarly, the NC330 cloned probe hybridized with the RT-PCR products from RNA of the induced-resistant tissue only. In Southern blot hybridization the NC330 DNA probe detected several genomic DNA fragments in both N. tabacum cv. Samsun NN and Samsun nn. The size of the DNA fragments differed in Samsun NN and Samsun nn. We suggest that DNA encoding the IVR-like protein is present in resistant and susceptible N. tabacum genotypes, but is expressed only in NN. We have inserted the NC330 into the expression vector pET22b and a 21.6 kDa protein was produced in Escherichia coli that reacted in immunoblots with the IVR antibody. This protein greatly reduced replication of TMV in N. tabacum cv. Samsun nn leaf disk assays.

Relationships between Leaf Age, Peroxidase and β‐1,3‐GIucanase Activity, and Resistance to Downy Mildew in Grapevines

The susceptibility of leaves of various ages of field and greenhouse-grown grapevines (Vitis vinifera), to downy mildew caused by Plasmopara viticola was evaluated. Older leaves from the bottoms of the shoots were more resistant to natural infection in the field or to artificial inoculation in the growth chamber with P. viticola, than were younger leaves from the tops of the shoots. This age-related resistance was confirmed by the use of quantitative leaf disc assay. The areas of lesions and sporangia produced on leaf discs obtained from leaves of various ages decreased significantly with increasing leaf age. This resistance was related to increases in β-1,3-glucanase and peroxidase activities, as determined on the same leaves from which leaf discs were obtained. Remarkable increases of up to 10-30 times and two- to sevenfold in peroxidase were found in the soluble and ionically bound fractions, respectively, of older leaves, as compared with younger leaves. Lower increase of 87 and 65% in β-1,3-glucanase was found in the soluble and ionically bound fractions, respectively, of older leaves, as compared with younger leaves. The data suggest the presence of defence mechanisms against P. viticola in grapevine leaves, that appear to be dependent on leaf age and which may involve these enzymes.

Association of Mmd1, a Major Gene for Resistance to Melampsora medusae f. sp. deltoidae, with Quantitative Traits in Poplar Rust

ABSTRACT A single gene, Mmd1, which conditions resistance and a necrotic flecking response to a monouredinial isolate of Melampsora medusae f. sp. deltoidae and is presumed to possess the corresponding avirulence gene, was previously shown to segregate 3:1 (resistant to susceptible) in an interspecific hybrid poplar F(2) progeny. Some inoculated clones of the resistant phenotypic class of this progeny were completely resistant and bore no uredinia, but most bore some sporulating uredinia with accompanying necrotic flecking. The dominant allele at the Mmd1 locus in these incompletely resistant clones was significantly associated with reduced uredinial density and diameter and longer latent period in a growth-room assay and with reduced disease incidence and infection efficiency and longer latent period in a leaf-disk assay. However, high clone-mean heritabilities within the susceptible phenotypic class indicated that genes other than Mmd1 also contribute to control of quantitative traits. Leaf age had a significant effect on uredinial density and latent period but not on uredinial diameter in the growth-room assay. All quantitative traits were intercorrelated to varying extents. A principal components analysis (PCA) demonstrated that uncorrelated components associated with uredinial diameter and uredinial density explained two-thirds of the total variation. Since uredinial diameter (PC1) and necrotic flecking are the visual components of an infection-type rating scale, genetic analyses of poplar rust should be based on infection type. Mmd1 is a major gene for resistance associated with a significant effect on all quantitative traits measured. Gene complexes in domesticated, agricultural rust pathosystems known to provide durable resistance consist of similar "pivotal" major genes and inferred ancillary genes or quantitative trait loci.

Chlorophyll fluorescence assay for kanamycin resistance screening in transgenic plants.

The applicability of a chlorophyll fluorescence assay for kanamycin (Km) resistance screening in transgenic tobacco (Nicotiana tabacum) and Arabidopsis thaliana plants was investigated. In wild-type leaves incubated in the presence of 200 mg/l Km, a decrease in maximum variable fluorescence ((Fv)m) and a significant increase in constant fluorescence (Fo) were observed. Using (Fv)m/Fo as a screening parameter, we were able to distinguish Km-treated samples from untreated samples within 4 days. This parameter was applied to Km resistance screening using tobacco plants transformed with the nptII gene via Agrobacterium. Among 74 shoots selected on medium containing 200 mg/l Km, 37 plants were scored as Km sensitive by the chlorophyll fluorescence assay. These 74 scorings proved to be accurate, as reconfirmed by (1) polymerase chain reaction amplification of the transgene, (2) enzymatic assay of neomycin phosphotransferase and (3) leaf disc assay. Using the chlorophyll fluorescence assay, we could also screen 3-week old Arabidopsis plants carrying the nptII gene. These results clearly demonstrate the reliability and efficiency of this nondestructive assay for Km resistance screening of transgenic plants.

Generation of Resistance to the Diphenyl Ether Herbicide, Oxyfluorfen, via Expression of the Bacillus subtilis Protoporphyrinogen Oxidase Gene in Transgenic Tobacco Plants.

In an effort to develop transgenic plants resistant to diphenyl ether herbicides, we introduced the protoporphyrinogen oxidase (EC 1.3.3.4) gene of Bacillus subtilis into tobacco plants. The results from a Northern analysis and leaf disc assay indicate that the expression of the B. subtilis protoporphyrinogen oxidase gene under the cauliflower mosaic virus 35S promoter generated resistance to the diphenyl ether herbicide, oxyfluorfen, in transgenic tobacco plants.

Differences in the rust resistance of greenhouse and outdoor‐grown white birch species, Betula spp.

SummaryThe 2‐year‐old seedlings of five different white birch species (Betula platyphylla, Betula papyrifera, Betula pubescens, Betula pendula (two types) and Betula resinifera x Betula pendula) grown both in a greenhouse and outdoors, were inoculated in a leaf disc assay with two different birch rust (Mel‐ampsoridium betulinum) isolates from B. pendula and B. pubescens. The resistance of these birch species varied significantly. Resistance to the B. pubescens rust isolate was not related to the resistance of the B. pendula rust isolate. The behaviour of a birch genotype grown in the greenhouse did not correspond to the behaviour of the same genotype grown outdoors.The outdoor growth environment greatly increased the contents of soluble proteins, rubisco, chloro‐phyll and nitrogen in the leaves of diploid birch species (B. platyphylla, B. pendula and B. resinifera x B. pendula). For tetraploid and pentaploid species (B. pubescens and B. papyrifera, respectively) there was no such clear difference in the leaf physiological status between the seedlings grown outdoors and in the greenhouse. The C:N ratio was higher for the greenhouse‐grown seedlings in all the birch species, but the difference was significant only with the diploid species. The incidence of rust in the birch species did not correlate with any of the leaf physiological parameters studied. The adaptability of birch genotypes to the environment in relation to their resistance to birch leaf rust is discussed.

Sensitivity profiles ofMycosphaerella graminicolaandPhytophthora infestanspopulations to different classes of fungicides

Prior to the use of fungicides, the baseline sensitivity of individuals in a pathogen population may already differ by a factor of 10 to 100 between the least and the most sensitive isolates. In Mycosphaerella graminicola populations, this factor, measured in vitro, was 5 to 20 for both the strobilurin analogue azoxystrobin (baseline) and the triazole cyproconazole which has been in use for several years. In Phytophthora infestans populations, this factor, measured in a leaf disc assay, was about 100 for azoxystrobin (baseline), up to 1000 for the cyanoacetamide cymoxanil and >10000 for the phenylamide oxadixyl; both of the latter have been used for many years. In M. graminicola, cross-sensitivity was present between all azole fungicides for the majority of the isolates, whereas no correlation was found between triazoles and azoxystrobin. Despite the existence of cross-sensitivity between azoles, ‘box-and-whiskers’ plots revealed large variations in the sensitivity profiles of some triazoles; isolates resistant to triazoles have not been detected in M. graminicola populations. In P. infestans populations, the proportion of the phenylamide-resistant sub-population increased during the season more rapidly in treated than in untreated fields, but it was low at the beginning of the next season in all fields. During disease epidemics, the fitness of phenylamide-resistant P. infestans isolates, as characterised by lesion size, was higher than that of the sensitive isolates, but after the overwintering period, the recovery of resistant isolates was apparently lower. The presence of both A1 and A2 mating types of P. infestans in European populations, although at different frequencies, allows sexual recombination and increased genetic diversity, affecting sensitivity and fitness. Such mixed populations can still be adequately controlled by using sound anti-resistance strategies. ©1997 SCI

A mutation in the 14 alpha-demethylase gene of Uncinula necator that correlates with resistance to a sterol biosynthesis inhibitor.

We investigated the molecular basis of resistance of the obligate biotrophic grape powdery mildew fungus Uncinula necator to sterol demethylation-inhibiting fungicides (DMIs). The sensitivity of 91 single-spore field isolates of U. necator to triadimenol was assessed by using a leaf disc assay. Resistance factors (RF) ranged from 1.8 to 26.0. The gene encoding the target of DMIs (eburicol 14 alpha-demethylase) from five sensitive and seven resistant isolates was cloned and sequenced. A single mutation, leading to the substitution of a phenylalanine residue for a tyrosine residue at position 136, was found in all isolates exhibiting an RF higher than 5. No mutation was found in sensitive or weakly resistant (RF, < 5) isolates. An allele-specific PCR assay was developed to detect the mutation. Among the 91 isolates tested, only isolates with RF higher than 5 carried the mutation. Three of the 19 resistant isolates and all sensitive and weakly resistant isolates did not possess the mutation. The mutation at codon 136 is thus clearly associated with high levels of resistance to triadimenol.

Behavioral and Sublethal Effects of Structurally Related Lower Terpenes on Myzus persicae

The sesquiterpenes farnesol, caryophyllene oxide, and 2,10-bisaboladien-1-one (patent #9602748) and the monoterpenes citronellol and geraniol were tested for settling inhibition and chronic effects on the aphidMyzus persicae by means of an improved leaf-disk assay. Of these compounds, geraniol, famesol, and the natural bisabolene significantly inhibit settling in choice tests. Furthermore, application of the bisabolene to intactCapsicum annuum leaves did not cause phytotoxicity, but did affect the insects' probing behavior by decreasing the probing activity and the number of intracellular punctures. Both compounds significantly decreased offspring production. Of the compounds tested, the natural product bisabolene could be a promising lead for future development of aphid control agents.

Effects of Water Stress on Colonization of Poplar Stems and Excised Leaf Disks by Septoria musiva

ABSTRACT Septoria musiva causes leaf spot and canker diseases of trees in the genus Populus, and is one of the most damaging fungal pathogens of hybrid poplar in eastern North America. The effect of host water stress on Septoria canker development was studied in two separate greenhouse experiments. Hybrid poplar clones NM6, NC11396, and NE308 were stressed by withholding water until predawn water potential fell below -1.0 MPa. Stems were treated by removing a leaf and applying agar plugs that were either colonized by S. musiva (inoculated) or sterile (control) to the wound. Cankers on inoculated water-stressed trees were significantly larger than those on nonstressed trees. A leaf disk assay also was conducted three times with the NM6 and NE308 trees. We cut two disks from each of 120 stressed and 120 well-watered trees, placing them on water agar in 24-well tissue culture plates. A conidial suspension was applied to one disk in each pair and sterile water to the other. Inoculated disks from water-stressed trees developed less necrosis than those from well-watered trees. These results demonstrate that environmental influences on host condition must be considered in evaluating resistance of clones proposed for widespread culture of hybrid poplar.