Sensitivity of Phytophthora infestans to mandipropamid and the effect of enforced selection pressure in the field

Sensitivity to the new carboxylic acid amide (CAA) fungicide mandipropamid (MPD) in Phytophthora infestans was measured for isolates collected between 1989 and 2002 in Israel prior to the commercial use of MPD (baseline sensitivity, 44 isolates), and from MPD-treated (25 isolates) and untreated fields (215 isolates) in nine European countries and Israel between 2001 and 2005. All isolates were sensitive to MPD, with EC₅₀ values ranging between 0·02 and 2·98  µ g mL⁻¹. Plastic-tunnel (UK), shade-house (Israel) and field experiments (Israel) conducted during 2001–05 showed that enforced selection pressure, applied preventively or curatively, imposed by repeated sublethal (5  µ g mL⁻¹) or excessive (500–1000  µ g mL⁻¹) doses of MPD on mixed isolates of P. infestans produced no isolates resistant to the compound. The results of this study indicate that the probability of a buildup of resistant sub-populations of P. infestans to mandipropamid in the field is low.     

Sensitivity of Sclerotinia homoeocarpa to demethylation-inhibiting fungicides in Ontario, Canada, after a decade of use

In late 2003, nine populations of Sclerotinia homoeocarpa in Ontario Canada (seven of which had been previously sampled in early 1994, prior to the registration of sterol demethylation-inhibiting (DMI) fungicides for turf disease control in Canada) were sampled and tested for sensitivity to propiconazole. Four of the nine populations had not been treated with DMI fungicides during the intervening years, and isolates from these locations were sensitive to propiconazole (geometric mean EC₅₀ values of 0·005–0·012 µ g mL⁻¹, compared with 0·005–0·008 µ g mL⁻¹ for the original 1994 populations). Among the five populations from 2003 that had been exposed to DMI fungicides, mean EC₅₀ values were significantly greater, ranging from 0·020 to 0·048 µ g mL⁻¹. A significant correlation of determination was found between estimated number of fungicide applications and log EC₅₀ ( R ² = 0·832, P  = 0·0001), and the equation predicted that 42·3 applications of propiconazole would be needed to bring a sensitive population (EC₅₀ < 0·01  µ g mL⁻¹) to a resistant level (EC₅₀ > 0·10  µ g mL⁻¹). Fungicide sensitivity vs. duration of fungicide efficacy was also tested, and it was found that isolates with decreased sensitivity were able to more quickly overcome the inhibitory effects of fungicide application, reducing the duration of control from 3 weeks to 2 weeks.     

Metalaxyl sensitivity of isolates of Phytophthora erythroseptica in Prince Edward Island

Isolates of Phytophthora erythroseptica , causal agent of pink rot of potato, were obtained from diseased tubers collected in Prince Edward Island (PEI), Canada. In an in vitro assay of field and single-zoospore isolates, all isolates of the pathogen from PEI were sensitive to metalaxyl (EC₅₀ < 0·5  µ g mL⁻¹), unlike moderately or highly resistant reference isolates obtained from Maine, USA. Allozyme-banding patterns at the glucose-6-phosphate isomerase ( Gpi ) locus were identical (91/91) for all isolates of P. erythroseptica examined from PEI and Maine, but could be used to distinguish isolates of P. erythroseptica from local isolates of Phytophthora infestans . Inoculation of potato tubers from plants treated with foliar applications of metalaxyl in the field indicated that compounds inhibitory to metalaxyl-sensitive isolates of P. erythroseptica were present in the periderm, even after 4 months' storage. By contrast, moderately or highly resistant isolates of the pathogen caused significantly ( P  ≤ 0·05) more disease than sensitive isolates in tubers from plants treated with metalaxyl. The effectiveness of metalaxyl in controlling pink rot in a particular region will depend on resistance levels of local populations of P. erythroseptica . Preventing the development and spread of resistant strains of P. erythroseptica will be critical in maintaining metalaxyl as an effective chemical for control of this pathogen.     

Reduced sensitivity of Cercospora beticola isolates to sterol-demethylation-inhibiting fungicides

In a survey conducted during October 1995, single-lesion isolates of the sugar beet leaf-spot fungus, Cercospora beticola , were tested for sensitivity to the sterol demethylation inhibiting fungicides (DMIs) flutriafol and bitertanol. The isolates were collected from fields in three different areas of northern Greece. Fields at Serres and Imathia had been sprayed with DMIs for about 15 years to control sugar beet leaf-spot. At the third site, Amyndeon, DMI fungicides had not been used. From each area 150 isolates were tested. ED₅₀ values were calculated for individual isolates by regressing the relative inhibition of colony growth against the natural logarithm of the fungicide concentration. The mean ED₅₀ values for flutriafol for the Serres, Imathia and Amyndeon populations were 1·07, 0·73 and 0·5 µg mL⁻¹, respectively (significantly different at P  = 0·05). For bitertanol the mean ED₅₀ values for the Serres and Imathia populations were 0·72 and 0·81 µg mL⁻¹, respectively, which were not significantly different at P  = 0·05. The mean ED₅₀ value of the Amyndeon population was 0·48 µg mL⁻¹, which was significantly lower than those of the other two populations ( P  < 0·05). A cross-resistance relationship was found to exist between the two triazole fungicides tested when log transformed ED₅₀ values of 60 isolates were subjected to a linear regression analysis ( r  = 0·81).     

Occurrence of Corynespora cassiicola isolates resistant to boscalid on cucumber in Ibaraki Prefecture, Japan

A total of 651 isolates of cucumber corynespora leaf spot fungus ( Corynespora cassiicola ) collected from cucumber in Japan, either with (438 isolates) or without (213 isolates) a prior history of boscalid use, were tested for their sensitivity to boscalid by using a mycelial growth inhibition method on YBA agar medium. Additionally, seven isolates of C. cassiicola obtained from tomato, soybean, eggplant (aubergine) and cowpea in different locations in Japan were tested before boscalid registration. Minimum inhibitory concentration (MIC) and 50% effective concentration (EC₅₀) values for 220 isolates from crops without a prior history of boscalid use ranged from 0·5 to 7·5 μg mL⁻¹ and from 0·04 to 0·59 μg mL⁻¹, respectively. Two hundred and fourteen out of 438 isolates collected from ten cucumber greenhouses in Ibaraki Prefecture, Japan, which received boscalid spray applications showed boscalid resistance, with MIC values higher than 30 μg mL⁻¹. Moreover, resistant isolates were divided into two groups: a moderately resistant (MR) group consisting of 189 isolates with EC₅₀ values ranging from 1·1 to 6·3 μg mL⁻¹, and a very highly resistant (VHR) group consisting of 25 isolates with EC₅₀ values higher than 24·8 μg mL⁻¹. MR isolates were detected from all ten greenhouses, but VHR isolates were detected from only three. As a result of fungus inoculation tests which used potted cucumber plants, control failures of boscalid were observed against resistant isolates. Efficacy of boscalid was remarkably low against VHR isolates in particular. This is the first known report on boscalid resistance in Japan.     

Biological control of fusarium crown and root rot of tomato with antagonistic bacteria and integrated control when combined with the fungicide carbendazim

Two bacterial isolates, Bacillus megaterium (c96) and Burkholderia cepacia (c91), demonstrated to be antagonistic against Fusarium oxysporum f.sp. radicis-lycopersici , the causal organism of fusarium crown and root rot of tomato, were evaluated as biocontrol agents alone and when integrated with the fungicide carbendazim. In an initial screening, these isolates reduced disease incidence by 75 and 88%, respectively. In vitro , both biocontrol agents were highly tolerant to the fungicide carbendazim, commonly used to control fusarium diseases. Carbendazim reduced disease symptoms by over 50% when used at > 50  µ g mL⁻¹, but had little effect at lower concentrations. Combination of the bacterial isolates and carbendazim gave significant ( P  ≤ 0·05) control of the disease when plants were artificially inoculated with the pathogen. Application of carbendazim at a low concentration (1  µ g mL⁻¹) in combination with B. cepacia c91 reduced disease symptoms by 46%, compared with a reduction of 20% obtained with the bacterium alone and no control with the chemical treatment alone. A combination of B. megaterium c96 with an increased application rate of 10  µ g mL⁻¹ carbendazim significantly reduced disease symptoms by 84% compared with inoculated controls and by 77% compared with carbendazim treatment alone. In this experiment, the integrated treatment also slightly outperformed application of 100  µ g mL⁻¹ carbendazim, and bacteria applied without fungicide also provided good disease control.     

Management of late leaf spot of groundnut (Arachis hypogaea) with chlorothalonil-tolerant isolates of Pseudomonas aeruginosa

Fifteen groundnut-associated bacterial isolates that inhibited by > 90% the in vitro conidial germination of Phaeoisariopsis personata , causal agent of late leaf spot disease of groundnut, were applied as a prophylactic spray (10⁸ cfu mL⁻¹) and tested for control of the disease in the glasshouse. Two groundnut seed-associated bacterial isolates, GSE 18 and GSE 19, identified as Pseudomonas aeruginosa , reduced the lesion frequency (LF) by up to 70%. A 90-day-old peat-based formulation of P. aeruginosa GSE 18 reduced LF measured 15 days postinoculation by up to 60%. Both P. aeruginosa GSE 18 and GSE 19 were tolerant to chlorothalonil (Kavach®) up to 2000  µ g mL⁻¹ in LB broth. In glasshouse trials, GSE 18 and GSE 19 tested in combination with reduced concentrations of chlorothalonil were highly efficient in management of the disease. The disease was completely controlled by chlorothalonil (> 250  µ g mL⁻¹), and in the presence of GSE 18 or GSE 19, 100  µ g mL⁻¹ chlorothalonil was equally effective. Application of rifamycin-resistant mutants of GSE 18 or GSE 19 together with chlorothalonil significantly increased the survival of these isolates in the groundnut phylloplane. In the field, a combination of GSE 18 and 500  µ g mL⁻¹ chlorothalonil reduced disease severity comparable to 2000  µ g mL⁻¹ chlorothalonil alone. Use of chlorothalonil-tolerant pseudomonads together with a quarter concentration of the recommended field dose of chlorothalonil doubled pod yield compared with the untreated unsprayed control.     

Biological diversity of Rhizoctonia solani (AG-3) in a northern potato-cultivation environment in Finland

A total of 119 isolates of Rhizoctonia were collected from stem canker lesions, stolon and root lesions, hymenia on stems, or from black scurf on tubers of potato plants ( Solanum tuberosum ) in Finland (latitudes 60–67°N). All isolates except three belonged to anastomosis group 3 (AG-3) of R. solani , as determined by phylogenetic analysis of the internal transcribed spacer sequences (ITS1 and ITS2) of ribosomal RNA (rRNA) genes. Sensitivity of the 119 isolates to the fungicide flutolanil was tested in vitro (EC₅₀ values 0·14–0·75  µ g active ingredient mL⁻¹). The isolates also varied considerably in growth rate (5·1–14·8 mm day⁻¹). The severity of disease caused by 99 isolates was determined based on the proportion of potato sprouts affected by lesions, discoloration or death, which was c . 1–60%. Only two isolates that were able to cause severe symptoms showed particularly low sensitivity to the fungicide and rapid growth rate. One isolate each of anastomosis groups AG-2-1 and AG-5 and an unknown, binucleate Rhizoctonia sp. were detected. The AG-5 isolate and the binucleate isolate caused mild symptoms on potato sprouts, whereas the AG-2-1 isolate was not pathogenic. Taken together, AG-3 of R. solani was the predominant causal agent of the stem canker and black scurf diseases of potato in Finland and showed considerable variability in disease severity, fungicide sensitivity and growth rate in vitro .     

Resistance of Pseudoperonospora cubensis to flumorph on cucumber in plastic houses

Plastic-house experiments were conducted over a 2-year period (2004–05) to estimate the effects of successive applications of flumorph or a mixture of flumorph with mancozeb to cucumber plants on selection for flumorph resistance in the downy mildew oomycete, Pseudoperonospora cubensis . Application of flumorph alone favoured the selection of resistant isolates of Ps. cubensis . Resistant populations were detected at a frequency of 2·5% after six successive applications of flumorph alone in a plastic house in 2004. Resistant isolates were also detected (4·8%) after eight successive applications of the mixture of flumorph and mancozeb in 2004, although the mixture gave significantly better disease control than flumorph alone and produced a slight delay in the development of resistance. In a second cucumber crop in the same plastic houses in 2004, the frequency of resistant isolates increased to 100% after three successive applications of flumorph or four of flumorph + mancozeb. Under laboratory conditions, most flumorph-resistant isolates showed high levels of resistance and their levels of pathogenicity and sporulation were as high as that of wild-type isolates. Flumorph showed cross-resistance with dimethomorph and iprovalicarb, but not with azoxystrobin, cyazofamid, cymoxanil or metalaxyl. These studies suggest a high risk for the occurrence of resistance to flumorph in Ps. cubensis in cucumber crops under plastic-house conditions.     

Sensitivity of Botrytis cinerea from vegetable greenhouses to boscalid

Between 2004 and 2006, 228 isolates of Botrytis cinerea from two regions in China were characterized for baseline sensitivity to boscalid, a new active ingredient that interferes with succinate ubiquinone reductase in the electron transport chain. The isolates showed similar sensitivity in different years and regions. Baseline sensitivities were distributed as unimodal curves with mean EC₅₀ values of 1·07 (± 0·11) and 0·42 (± 0·05) mg L⁻¹ for inhibition of mycelial growth and conidial germination, respectively. Laboratory studies were conducted to evaluate the risk of development of resistance to boscalid. Boscalid-resistant mutants were obtained by UV-treatment at lower frequencies and with smaller resistance factors than pyrimethanil-resistant mutants. All boscalid-resistant mutants were also significantly more sensitive to Qo inhibitors than their wild-type parents and showed reduced sporulation in vitro and pathogenicity on aubergine leaves. The results suggested that the risk of resistance developing for boscalid was lower than for pyrimethanil. However, as B. cinerea is a high-risk pathogen, appropriate precautions against resistance development should be taken. Synergism between the activity of boscalid and that of kresoxim-methyl was observed.     

Control of Phytophthora cryptogea in the hydroponic forcing of witloof chicory with the rhamnolipid-based biosurfactant formulation PRO1

Rhamnolipids, extracellular metabolites of Pseudomonas aeruginosa with surfactant properties, proved to be very effective in controlling the spread of brown root rot disease caused by Phytophthora cryptogea in the hydroponic forcing system of witloof chicory ( Cichorium intybus var. foliosum ). The biosurfactant was applied as the product PRO1, a formulation of 25% rhamnolipids in oil. Both an in vitro screening and in vivo experiments in a mini-hydroponic system demonstrated the ability of PRO1 to control brown root rot. A 25  µ g mL⁻¹ rhamnolipids nutrient solution was enough to obtain good control of an artificial infection with a zoospore suspension of P. cryptogea . The biosurfactant PRO1 performed well in a semicommercial system under growers' conditions. A treatment of 25  µ g mL⁻¹ rhamnolipids (100  µ g mL⁻¹ PRO1) reduced the disease incidence significantly in two independent experiments. However, PRO1 was not effective when a mycelial suspension was used as inoculum. Rhamnolipids have good potential to limit the spread of P. cryptogea in the hydroponic forcing system of witloof chicory, and can be used as a preventive measure against brown root rot.     

Effect of metalaxyl on formation and germination of oospores of Phytophthora infestans

Oospores of Phytophthora infestans were produced in potato leaf discs floating on metalaxyl solution (100 μg mL⁻¹ a.i.) and inoculated with all combinations of two metalaxyl-sensitive and two -resistant parental isolates. Numbers of oospores produced varied between different matings, depending on parents, in the absence of the fungicide and when metalaxyl was added 0, 7, 14 and 21 days after inoculation. Oospores were not produced when metalaxyl was added at the time of inoculation (0 days) when either one or both parents were sensitive to metalaxyl. In two of three such matings further oospore formation was arrested when metalaxyl was added either 7 or 14 days after inoculation. Oospores extracted from leaf discs 14, 21 and 28 days after inoculation were assessed for germination on water agar after 21 days. Germination of oospores from water control treatments varied between 6 and 30% depending on the cross. Germination was significantly reduced in oospores of metalaxyl-sensitive parents extracted 28 days after inoculation of leaf discs treated with metalaxyl 0, 7 and 14 days after inoculation compared with the 21-day treatment. Minimal differences in germination were observed for oospores from the mating of resistant parents irrespective of metalaxyl treatment, although germination was generally low, not exceeding 8.5%.     

Fungicide sensitivity in Swedish isolates of Phaeosphaeria nodorum

This is the first report of variability in sensitivity of Phaeosphaeria nodorum to the fungicide azoxystrobin, and also reports on sensitivity to propiconazole, prothioconazole and cyprodinil. An in vitro sensitivity test of 42 isolates from five Swedish winter wheat fields, collected in 2003–2005, was performed on malt extract agar amended with six concentrations of each fungicide. Four isolates collected during the early 1990s, before azoxystrobin had been commercially used in agriculture, were used as references. Fragments of DNA from 231 isolates, including the reference isolates, were sequenced for the genes of cytochrome b and CYP51 in search for amino acid substitutions known to cause loss of sensitivity to strobilurins and triazoles, respectively. The majority of the P. nodorum isolates possessed the amino acid substitution G143A, associated with loss of sensitivity in fungi to strobilurins, except in one field where only half of the isolates had the substitution. The EC₅₀ values varied between 0·66 mg L⁻¹ to estimations far above 1000 mg L⁻¹, with an estimated median value of 366 mg L⁻¹. The EC₅₀ values of the reference isolates ranged from 0·02 to 80·72 mg L⁻¹. The P. nodorum population is still sensitive to propiconazole, prothioconazole and cyprodinil, even though some isolates varied in sensitivity to triazoles. Part of the CYP51 gene, a possible target for triazole sensitivity, was sequenced but no nonsynonymous substitutions were found.     

In vitro antifungal activity of brassinin, camalexin and two isothiocyanates against the crucifer pathogens Alternaria brassicicola and Alternaria brassicae

In vitro assays investigated the responses of Alternaria brassicicola and A. brassicae isolates towards two crucifer phytoalexins and two isothiocyanates (ITC) by evaluating their potential toxic effects on different fungal growth parameters. Although variable responses towards each compound was observed within the species A. brassicicola , the results obtained confirmed the antifungal effects of camalexin, brassinin, allyl- (AlITC) and benzyl- (BzITC) isothiocyanates, at different developmental stages of both Alternaria species. Irrespective of the tested isolate, the phytoalexin camalexin exhibited the greater inhibitory effect with mean EC₅₀ values ranging from 34  µ m (germ-tube elongation) to 183  µ m (mycelial growth). Germ-tube elongation was more sensitive compared to conidial germination and mycelial growth, with mean EC₅₀ values of the former of 81  µ m , 520  µ m and 870  µ m for brassinin, BzITC and AlITC, respectively.     

Sensitivity of fungi from cereal roots to fluquinconazole and their suppressiveness towards take-all on plants with or without fluquinconazole seed treatment in a controlled environment

The take-all fungus, Gaeumannomyces graminis var. tritici , was highly sensitive to fluquinconazole ( in-vitro EC₅₀ 0·016–0·018 mg L⁻¹), a fungicide developed for use as a seed treatment to control take-all, and to prochloraz (EC₅₀ 0·006 mg L⁻¹). Fungi of other genera that were commonly isolated from cereal roots were sensitive in varying degrees to prochloraz but were relatively insensitive (e.g. Fusarium culmorum , EC₅₀ > 20 mg L⁻¹) or slightly sensitive (e.g. Epicoccum purpurascens , EC₅₀ 0·514 mg L⁻¹) to fluquinconazole. Gaeumannomyces graminis var. graminis and G. cylindrosporus , weak parasites that can protect roots against take-all, and an unnamed Phialophora sp., all closely related to the take-all fungus, were highly or moderately sensitive to fluquinconazole. Alternaria infectoria and E. purpurascens were most consistently effective in suppressing development of take-all on pot-grown wheat plants dual-inoculated with G. graminis var. tritici and the nonpathogen. Take-all was decreased more on dual-inoculated wheat plants grown from seed treated with fluquinconazole or fluquinconazole plus prochloraz than when only an antagonistic fungus ( A. infectoria , E. purpurascens , Fusarium culmorum or Idriella bolleyi ) or a seed treatment was applied. These fungi were less effective in combination with seed treatments on barley. Gaeumannomyces graminis var. graminis and G. cylindrosporus , tested on wheat, suppressed take-all only in the absence of fungicides. It is suggested that the performance of seed treatment containing fluquinconazole against take-all may in some circumstances be enhanced by its partial specificity for the take-all fungus.     

Sulphur accumulation after Verticillium dahliae infection of two pepper cultivars differing in degree of resistance

Elemental sulphur levels, sulphur localization in stems, and levels of sulphate, glutathione and cysteine were studied in pepper ( Capsicum annuum ) cvs Yolo Wonder (higher resistance) and Luesia (lower resistance) after inoculation with Verticillium dahliae , the cause of vascular wilt. Accumulation of elemental sulphur (S⁰) was first detected 10 days after inoculation in Yolo Wonder (mean S⁰ level 7·3  µ g g⁻¹ DW), and 15 days after inoculation in Luesia (mean S⁰ level 3·3  µ g g⁻¹ DW). The maximum level was reached 21 days after inoculation in Yolo Wonder (14·1  µ g g⁻¹ DW). In control plants, elemental sulphur was not detected. SEM-EDX (scanning electron microscopy-energy dispersive X-ray microanalysis) indicated that the sulphur was not restricted to a specific location, but was dispersed throughout the vascular tissue. Sulphate levels showed a decline at the end of the experiment in inoculated plants, possibly related to the increase in sulphur levels seen in the two cultivars. The differences in sulphate levels between the two cultivars may be due to faster sulphate breakdown in cv. Yolo Wonder.     

Aggressiveness and host specificity of Brazilian isolates of Phytophthora infestans

The population of Phytophthora infestans in Brazil consists of two clonal lineages, US-1 associated with tomatoes and BR-1 associated with potatoes. To assess whether host specificity in these lineages resulted from differences in aggressiveness to potato and tomato, six aggressiveness-related epidemiological components – infection frequency (IF), incubation period (IP), latent period (LP), lesion area (LA), lesion expansion rate (LER) and sporulation at several lesion ages (SSLA) – were measured on detached leaflets of late blight-susceptible potato and tomato plants. Infection frequency of US-1 was similar on potato and tomato leaflets, but IF of BR-1 was somewhat reduced on tomato. Incubation period was longer on both hosts with US-1, although this apparent lineage affect was not significant. Overall there was no host effect on IP. On potato, BR-1 had a shorter LP (110·3 h) and a larger LA (6·5 cm²) than US-1 (LP = 162·0 h; LA = 2·8 cm²). The highest LER resulted when isolates of BR-1 (0·121 cm² h⁻¹) and US-1 (0·053 cm² h⁻¹) were inoculated on potato and tomato leaflets, respectively. The highest values of the area under the sporulation capacity curve (AUSC) were obtained for isolates of US-1 inoculated on tomato leaflets (6146) and for isolates of BR-1 on potato leaflets (3775). In general, higher values of LA, LER, SSLA and AUSC, and shorter values of LP were measured when isolates of a clonal lineage were inoculated on their original host than with the opposite combinations. There is evidence that there are quantitative differences in aggressiveness components between isolates of US-1 and BR-1 clonal lineages that probably contribute to host specificity of P. infestans populations in Brazil.     

Resistance to Sphaerotheca pannosa in roses induced by 2,6-dichloroisonicotinic acid

The susceptible rose cv. Madelon and the partially resistant cv. Sonia both responded with reduced development of rose powdery mildew when they were treated with the synthetic inducer 2,6-dichloroisonicotinic acid (INA). The EC₅₀ for number of colonies cm⁻² was approximately 0.4 mg L⁻¹ in both cultivars when treated 4 days prior to inoculation. However, conspicuous differences were observed with respect to number of spores per cm². For sporulation, the EC₅₀ was 0.37 mg L⁻¹ in cv. Madelon and only 0.08 mg L⁻¹ in cv. Sonia. A comparison with the pathosystems cucumber/ Sphaerotheca fuliginea and red cabbage/ Peronospora parasitica is made and the importance of the observed phenomenon for the selection of parents in a breeding programme for (partial) resistance is discussed.     

In vitro somatic growth and reproduction of phenylamide-resistant and -sensitive isolates of Phytophthora erythroseptica from infected potato tubers in Idaho

Pink rot of potato, most commonly caused by Phytophthora erythroseptica , is a major field and post-harvest problem in southern Idaho, USA, particularly since 1998 when isolates resistant to the phenylamide fungicide metalaxyl-M (mefenoxam) were detected. Isolates of P. erythroseptica were collected from infected tubers in 2001 and 2002 from six Idaho counties and tested for resistance to metalaxyl-M on amended agar. Metalaxyl-M resistant (MR) and metalaxyl-M-sensitive (MS) isolates were identified in six counties; 160 isolates were highly resistant, seven moderately resistant and 57 sensitive to metalaxyl-M with mean EC₅₀ values of 182, 23 and 0·5 mg L⁻¹ ai metalaxyl-M, respectively. Mycelial growth rates and oospore production in agar were assessed for 20 MS and 20 MR isolates at 10, 15, 20, 25 and 30°C. Growth rates of MR isolates were between 2·5 and 3·1 times greater ( P  < 0·05) than those of MS isolates at 10, 15, 20 and 25°C, and oospore production was between 6·8 and 20·5 times greater ( P  < 0·0001) for MR than for MS isolates at the same temperatures. Colony growth in V8 broth at 18°C was greater for MR than MS isolates ( P  < 0·0032). However, zoospore production at 18°C was greater for MS than for MR isolates ( P  < 0·0109), and zoospore production m m ⁻¹ of colony circumference was also greater for MS than for MR isolates, 14 191 and 9959, respectively ( P  = 0·0109). Sexual reproduction of MR isolates in nature may be greater than MS isolates, but MS isolates may be more asexually fit based on the fitness parameters studied.     

Nematicidal activity of essential oils and organic amendments from Asteraceae against root-knot nematodes

The essential oil of Chrysanthemum coronarium flowerheads showed strong nematicidal activity in vitro and in growth-chamber experiments. Essential oil concentrations of 2, 4, 8 and 16  µ L mL⁻¹, significantly reduced hatch, J ₂ survival (determined by final value and area under curves of cumulative percentage hatch or mortality) and reproduction rate of Meloidogyne artiellia in vitro , with the lowest values occurring at 16  µ L mL⁻¹. In pot trials with chickpea cv. PV 61, essential oil concentrations of 10–40  µ L per 500 cm³ soil, applied on sterile cotton pellets, also significantly reduced the nematode's reproduction rate. The biological processes of mortality and hatching/reproduction were adequately described by the monomolecular and expanded negative exponential models, respectively. Effectiveness of soil amendment with either flowers, leaves, roots or seeds of C. coronarium , and flowers from several species of Asteraceae ( Chrysanthemum segetum , Calendula maritima , Calendula officinalis and Calendula suffruticosa ) at 5 g per 500 cm³ soil was tested for suppression of M. artiellia and growth of chickpea cv. PV 61 under growth-chamber conditions. In these tests, flowers of all five Asteraceae species and various parts of C. coronarium significantly reduced reproduction rates of M. artiellia , by 83·0–95·9%, with the minimum rates occurring in infected chickpea plants amended with flowers of C. officinalis and C. suffruticosa . The in vitro and in planta results suggest that the essential oil of C. coronarium and organic amendments from Asteraceae species may serve as nematicides.