Effect of root-knot nematodes on cajanol accumulation in the vascular tissues of pigeonpea after stem inoculation with Fusarium udum

The effect of root-knot nematodes ( Meloidogyne javanica and M. incognita ) on production of the isoflavonoid phytolexin, cajanol, was investigated in pigeonpea ( Cajanus cajan ) plants infected with Fusarium udum , the causal organism of Fusarium wilt. Seven-day-old seedlings of a wilt-resistant pigeonpea cultivar, ICP 9145 and a wilt-susceptible cultivar, Malawi Local, both of which were moderately susceptible to the nematode, were grown in soil infested with 2000 Meloidogyne juyeniles per plant. A duplicate set of plants remained free from nematodes. Twenty-one days later, all the plants were inoculated with F. udum by stem puncture. Quantitative estimates of cajanol in the vascular tissues were made at intervals up to 15 days after inoculation with the fungus. No external symptoms of wilt appeared in any plants of the wilt-resistant cultivar in the absence of the nematode. However, when inoculated with the nematode, two thirds of the plants developed wilt symptoms. Cajanol levels were lower in both the wilt-resistant and wilt-susceptible plants in the presence of the nematode than in its absence, although this effect was considerably more marked in the wilt-resistant cultivar. These results indicate that the root-knot nematode is capable of breaking resistance of ICP 9145 to Fusarium wilt and that at least part of the mechanism of this effect is retarded cajanol accumulation.     

Induction of phytoalexins in pigeonpea (Cajanus cajan) in response to inoculation with Fusarium udum and other treatments

Cajanol accumulation was monitored in four cultivars of pigeonpea, Cajanus cajan (L) Millsp, after inoculation with 2 x 10(6) conidia ml-1 of Fusarium udum Butler. Rapid accumulation was observed only in wilt-resistant cultivar ICP 9145. Another wilt-resistant cultivar, ICP 8863, had low levels of cajanol similar to those in wilt-susceptible cultivars ICP 2376 and Malawi local, indicating that rapid cajanol accumulation is not positively correlated with resistance to wilt in pigeonpea. A comparison of various inducing agents showed live conidia of the pathogen to cause more rapid accumulation than the other agents.     

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.     

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.     

Antifungal activity of bergenin, a constituent of Flueggea microcarpa

The antifungal activity of bergenin against some plant pathogenic fungi, namely, Alternaria alternata , A. brassicae , A. carthami , Fusarium udum , F. oxysporum f.sp. ciceri , Curvularia lunata and Erysiphe pisi , was studied. Bergenin as its monosodium salt was effective against all the fungi and the effective dose for complete inhibition of spore germination varied from 15 μg mL⁻¹ for F. udum to 125 μg mL⁻¹ for E. pisi . Experiments on the effect of bergenin on powdery mildew development under glasshouse conditions revealed that it can control powdery mildew of pea at 2000 μg mL⁻¹ by postinoculation treatment, the results being comparable with those of carbendazim (1000 μg mL⁻¹) and wettable sulfur (2000 μg mL⁻¹). It affected hyphal elongation and the number of primary and secondary branches.     

Resistance to benzimidazole fungicides in the gummy stem blight pathogen Didymelta bryoniae on cucurbits

Benzimidazoles were used successfully against gummy stem blight of cucurbits for only one growing season (1980). It was soon noticed in experiments in vivo that they were not effective against some isolates of the pathogen. In a survey during the 1981 crop season, 76 out of 102 isolates collected from 20 plastic greenhouses all over the main cucurbit-growing areas of Crete were resistant to benomyl. The average benomyl concentrations that reduced mycelial growth rates of seven resistant and seven sensitive isolates by 50% were 716 ± 49 and 1.2 ± 0.34μg/ml respectively. The average growth rate of resistant isolates was lower than that of sensitive isolates but there were no differences in spore production and spore germination. The pathogenicity of resistant isolates on young cucumber plants was lower than that of sensitive isolates. The application of benomyl, carbendazim, cypendazole and thiophanate methyl to young cucumber plants at the rate of 300μg a.i./ml 1 day before inoculation with 500 000 spores/ml of the resistant isolates did not affect the infection rate of the disease after 4 days. Sensitive isolates caused no onslight symptoms on similarly treated plants.     

Variability in the reaction of squash (Cucurbita pepo) to inoculation with Sphaerotheca fuliginea and methodology of breeding for resistance

Reaction to inoculation with powdery mildew caused by Sphaerotheca fuliginea was observed on leaf discs and young plants of eleven representatives of seven edible cultivar groups of Cucurbita pepo. Disease intensity (i.e. number of infections per leaf) was highly correlated ( r ²=0·863, P <0·0001) with spore yield per leaf. Spore yield per leaf and frequency of sporulation on leaf disks were moderately ( r ²=0·505), but significantly correlated ( P <0·01), suggesting that frequency of sporulation can be used for initial screening against susceptibility in a breeding programme. Spore yield per leaf and spore yield per artificially inoculated leaf disc were highly correlated ( r ²=0·87, P <0·0001); this suggests that counting of spores on leaf discs, a laborious but accurate procedure, could be used on the remaining plants as a second step in selection for resistance of the variation in the response of edible C. pepo to the pathogen, 85·8% was attributed to differences between the edible groups and only 14·2% to individual cultivars within a group. Cultivars of the cocozelle and vegetable marrow groups were the most susceptible, whereas relatively resistant cultivars were found in the scallop and straightneck groups.     

A qualitative quick-test for detection of herbicide resistance to tribenuron-methyl in Papaver rhoeas

A qualitative seed-based method useful for the detection of resistance to the herbicide tribenuron-methyl in Papaver rhoeas L. is described. Seeds were germinated on 35 mL of a 1.3% agar medium containing 2 g KNO₃ L^–1 in 8.5 cm Petri dishes in a growth chamber under 20 μmol s^–1 m^–2 of fluorescent light. When 0.24 μM tribenuron-methyl or more was added, growth in susceptible plants stopped after the cotyledon stage and they turned chlorotic. The resistant plants continued developing new leaves. The same effect was achieved when 0.2 g gibberellin (GA₃) L^–1 and 7.68 μM tribenuron-methyl or 0.5 g GA₃ L^–1 and 61.44 μM tribenuron-methyl were added. Germination percentage rose with gibberellin in the presence or absence of the herbicide. Plants developed rapidly, with only about 14 d needed to finish the test but sometimes root growth was reduced because of the addition of gibberellin. In the absence of gibberellin but in the presence of the herbicide, plants grew more slowly and developed smaller leaves with a 17-d evaluation period requirement. The test was validated with pot experiments in a greenhouse and also with field trials. The best combination was found to be 0.2 g GA₃ L^–1 and 7.68 μM tribenuron-methyl, assuring homogenous germination and testing of dormant seeds but avoiding root inhibition associated with too much gibberellin.     

Effect of method of inoculation, inoculum density and seedling age at inoculation on the expression of resistance of cocoa (Theobroma cacao L.) to Verticillium dahliae Kleb.

Soil drench and stem puncture inoculation were compared as methods for selecting cocoa cultivars with resistance to Verticillium dahliae. Disease progress was more rapid and induced symptoms were more severe following stem puncture and, under glasshouse conditions, differences between cultivars were detected 15 days after inoculation. Moreover, using stem puncture, inoculum densities of 10⁴ conidia/ml were sufficient to differentiate resistant and susceptible cultivars, whereas with the soil drench method, inoculum densities of 10⁷ conidia/ml were necessary. Although a substantially higher proportion of plants were affected by stem puncture inoculation, the resistance of cultivar Pound-7 remained effective at high inoculum densities of 10⁸ conidia/ml. With either method, older seedlings were more susceptible to V. dahliae than younger ones. However, with stem puncture, 15-day-old seedlings were sufficiently susceptible for a valid disease assessment. In contrast, with soil inoculation, 60-day-old plants were required. In a nursery trial with 15-day-old seedlings, seven cocoa genotypes previously selected as resistant, moderately resistant or susceptible to Verticillium dahliae , on the basis of root inoculation, were ranked in the same order when stem punctured. Stem puncture inoculation of young seedlings is cost-effective in terms of time and space, and is therefore recommended for screening of cocoa for wilt resistance, especially in large-scale breeding programmes.     

Effects of avirulent bacteriocin-producing strains of Pseudomonas solanacearum on the control of bacterial wilt of tobacco

Root systems of tobacco dipped in suspensions containing 2 × 10⁹ colony forming units (CFU)/ml of avirulent bacteriocin-producing strains (ABPS) of Pseudomonas solanacearum and assayed immediately after planting in steam-sterilized soil had 8 × 10⁶ CFU/root system of ABPS. The bacterial population declined to an average of 5·3 × 10⁵ CFU/root system after 30 days. Roots of seedlings dipped in bacterial suspensions of ABPS were more effectively protected against wilt caused by P. solanacearum than those dipped in suspensions of an avirulent nonbacteriocin-producing strain (ANBPS). Lipopolysaccharide (LPS) isolated from one ABPS (121) inhibited the attachment of bacteria on roots by 70% but had no effect on the reduction of wilt, whereas bacterial cells significantly reduced the disease severity as compared to LPS or water treatment. In steam-sterilized soil containing a 1:1 mixture (5 × 10⁵ CFU/g of oven-dried soil) of ABPS 121 or 237 and the virulent strain K-60, ABPS 121 reduced multiplication of the virulent strain in soil and in the rhizosphere of seedlings. When roots of seedlings were dipped in a suspension of 2 × 10⁹ CFU/ml of ABPS before planting, root colonization by the virulent strain added to steam-sterilized soil at 2 × 10⁶ CFU/g of oven-dried soil was significantly reduced. When roots were dipped in a suspension of ABPS and assayed 20 days after planting, 98% of the bacterial population was found in the original zone of inoculation and only 2% was detected in new growths of the root system. Plants which were grown in soil infested with ABPS 121 or K-60 had both strains present at variable populations along all sections of roots.     

Slow wilting components in pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis>(L.) Millsp.)

Partial resistance to Fusarium wilt was characterized based on root inoculation on seven pigeonpea genotypes with a virulent isolate of Fusarium udum. Resistance to wilt seems to be mainly due to localization of the pathogen in roots and vascular systems in the stem. Three components of resistance (wilting rate or incubation period, weighted wilt index and number of colonized plants) were determined and compared to those of susceptible genotypes Bahar and TTB7. There were significant differences between the genotypes for the resistance components. The mechanisms of resistance in the genotypes appeared to be different, with genotype ICP8863, having a longer incubation period, minimum wilt index and minimum pathogen colonization as compared to other resistant genotypes (ICP9174, ICP87119 and ICP8858). Wilting rate or incubation period and number of colonized plant were significantly correlated with resistance in adult plants in the field (AUDPC). Wilt index was useful in discriminating between genotypes that had a similar incubation period and number of colonized plants. The partial character of resistance is probably based on quantitative differences in localization capacity among the genotypes. A quantitative relationship between components, incubation period and number of colonized plants and the AUDPC, if verified for a large number of genotypes, may be used to obtain an index of resistance that may predict resistance levels in the field.     

Control of Penicillium expansum by plant volatile compounds

Nine plant volatiles were tested for their activity in vitro and in vivo against Penicillium expansum , the cause of blue mould of pear. In vitro spore germination and mycelial growth assay showed a consistent fungicidal activity by trans -2-hexenal, carvacrol, trans -cinnamaldehyde and citral, while hexanal (-)- carvone, p -anisaldehyde, eugenol and 2-nonanone exhibited a progressively lower inhibition. trans -2-Hexenal was the best inhibitor of conidial germination [MIC (minimum inhibitory concentration) = 24·6  µ L L⁻¹; ED₅₀ = 10·2  µ L L⁻¹], while carvacrol was the best inhibitor of mycelial growth (MIC = 24·6  µ L L⁻¹; ED₅₀ = 9  µ L L⁻¹). The four most active compounds in in vitro studies were tested in vivo as fumigants against blue mould on pear cv. Conference. Best control was achieved by trans -2-hexenal vapour treatments (12·5  µ L L⁻¹) when applied over a 24-h period, beginning 24 h after inoculation. In contrast, carvacrol (12·5–200  µ L L⁻¹), and trans -cinnamaldehyde (50–400  µ L L⁻¹) were ineffective and citral (200  µ L L⁻¹) showed only slight effect.     

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%.     

Dicarboximide resistance in Botrytis cinerea in protected lettuce

In surveys of protected lettuce crops in the Humberside area of the UK during 1984-5, 47.3% of Botrytis cinerea isolates were resistant to dicarboximides and 25% to benomyl. Dicarboximide-resistant isolates obtained direct from the field had a moderate degree of resistance with ED₅₀ values for mycelial growth on agar of 1.0-2.4 μg/ml vinclozolin for all except two of 30 isolates.
Isolates which were resistant to vinclozolin were also resistant to iprodione, myclozolin and procymidone. Thirty dicarboximide-resistant isolates obtained direct from the field were capable of infecting cucumber cotyledons sprayed with field-strength Ronilan (250 μg/ml vinclozolin) whereas sensitive strains were not. Strains showing different degrees of resistance to dicarboximides developed after incubation of sensitive and resistant strains on agar supplemented with vinclozolin. Some of these strains had ED₅₀ values for mycelial growth on agar of more than 100 μg/ml vinclozolin. No highly resistant isolates were obtained direct from the field. Resistance to dicarboximides in field isolates was stable.     

儿茶素在棉苗对枯萎病抗性中的作用

 分析了枯萎病菌接种后棉苗组织内儿茶素含量的变化以及儿茶素对枯萎病菌及其多聚半乳糖醛酸酶活性的影响。健康棉苗组织中儿茶素含量随着棉苗的生长而增加。枯萎病菌接种后,病苗组织中儿茶素含量显著高于健康对照,而且抗病品种棉苗儿茶素含量的增加明显高于感病品种。经氟乐灵预处理的棉苗接种枯萎病菌后,儿茶素含量增加更快,增幅更大。说明儿茶素是品种抗性和氟乐灵诱发棉苗对枯萎病诱导抗性的机理之一。儿茶素可抑制枯萎病菌的菌丝生长、产孢及孢子萌发,对培养液中病菌的多聚半乳糖醛酸酶(PG)有抑制作用。儿茶素化学制品及棉苗组织提取液经TLC分离后获得的含儿茶素组分提取液对初步纯化的PG活性有明显的抑制作用。这些结果表明:儿茶素可能通过抑制病菌的菌丝生长、产孢以及其PG活性而在棉花对枯萎病的抗病性中起到作用。     

A new selective medium for Burkholderia caryophylli, the causal agent of carnation bacterial wilt

A new selective medium (APCA medium) was developed for the isolation of Burkholderia caryophylli , the causal agent of carnation bacterial wilt, from both plants and soil. The optimal concentration and combination of antibiotics was investigated to determine the most selective condition for growing B .  caryophylli . The resultant composition of the medium per litre was: 0·79 g (NH₄)₂SO₄, 1·0 g KH₂PO₄, 0·5 g MgSO₄ · 7H₂O, 0·2 g KCl, 2·0 g D-arabinose, 5 mg crystal violet, 50 mg cycloheximide, 50 mg polymyxin B sulphate, 50 mg ampicillin sodium, 10 mg chloramphenicol, 25 mg blue tetrazolium, and 15 g agar. Plating efficiency ranged from 119 to 174% with an average of 141% compared to that of nutrient agar. The bacterium was successfully isolated from contaminated soil and plant tissues with this medium. Moreover, the medium almost completely inhibited the growth of other plant pathogenic bacteria and soil saprophytes. This selectivity was high enough to detect B . caryophylli in contaminated soil.     

Controlled environment studies on the infection of Emex australis by Phomopsis emicis

The effect of temperature, dew period, inoculum concentration and host maturity on the growth and development of Emex australis after inoculation with Phomopsis emicis was studied in controlled environments. Disease was assessed by recording changes in the length of stems, number of fully expanded leaves, dry weights, number of fruits and disease severity index. Disease was as severe at 18°C as it was at 28°C. There was a trend of increased disease with longer dew period. Inoculum concentrations of 1·10⁶ and 1·10⁷ conidia per ml resulted in a significant net reduction in stem growth compared with lower concentrations. Progressively higher disease ratings occurred as the inoculum concentration increased to 1·10⁷ conidia per ml. There were no significant effects of inoculation with P. emicis on either seedlings, rosettes or flowering rosettes, but 10-week-old plants were highly susceptible to infection and stem collapse. The implications of these results for the potential development of P. emicis as a mycoherbicide are discussed.     

Analysis of resistance to Xylella fastidiosa within a hybrid population of Pera sweet orange × Murcott tangor

Resistance to Xylella fastidiosa was evaluated within a population of 20 interspecific hybrids of Pera sweet orange and Murcott tangor under greenhouse conditions. Efficiency of inoculation, multiplication of bacteria within the plants, xylem vessel morphology, and symptom expression were analysed. The rate of infection ranged from 40 to 100% (average 70%) for all genotypes analysed. Xylella fastidiosa populations ranged from log 0·59 to log 2·13 cells mg⁻¹ tissue for the resistant hybrids. These values were significantly different ( P  = 0·05) from those obtained for the tolerant (no symptoms but bacteria recovered) or susceptible (symptoms and bacteria recovered) hybrids (log 3·02 to log 4·06 cells mg⁻¹). Xylella fastidiosa was recovered from all hybrids (log 2·31 to 5·03 CFU mg⁻¹ tissue) except the resistant ones. The first foliar symptoms appeared at least 90 days post-inoculation, the time varying according to genotype. No correlation between xylem vessel morphology and disease expression was observed, indicating that the resistance was the result of a genetic response of the host. According to this hypothesis, a high broad-based heritability index for resistance was obtained (0·96) at 210 days from X. fastidiosa inoculations, using bacterial quantification by real-time PCR, which indicated that the influence of the number of bacteria was the result of genetic rather than environmental variations.     

Response of interspecific Brassica juncea/Brassica rapa hybrids and their advanced progenies to Albugo candida (white blister)

Transfer of factors for resistance to white blister disease caused by Albugo candida between Brassica species involving two genotypes each of B. juncea and B. rapa was studied in hybrids. More hybrids were obtained by in vivo than in vitro techniques, although an in vitro phase was a prerequisite for the establishment of in vivo hybrids. Hybrids were identified by PCR-based inter-simple sequence repeat (ISSR) markers with both male and female species-specific bands being identified. There was a positive correlation between disease severity and number of days after sowing ( r  > 0·93), the highest being towards pod formation and plant maturity at 110 days after sowing. The plants from F₂ and BC₁ progeny showed higher resistance to A. candida than either of the parents. Plants of B. juncea and B. rapa with high field resistance (disease index < 1·0) were selected from BC₂ and F₂BC₁ generations. The frequency of plants classified as resistant in BC₂ progeny ranged from 4·5 to 39·0% in cross-combinations involving B. juncea genotypes as female parent, compared with 100% in the reciprocal cross involving B. rapa as female parent.     

Reduced sensitivity of Botrytis cinerea to two sterol biosynthesis-inhibiting fungicides: fenetrazole and fenethanil

Isolates of Botrytis cinerea having reduced sensitivity to the sterol biosynthesis-inhibiting (SBI) fungicides fenetrazole and fenethanil were obtained from one out of four sites from which isolates were tested. Reduced sensitivity was associated with poor disease control by fenetrazole, which had been applied with dichlofluanid. Conidial germination and hyphal growth of B. cinerea from the four sites were tested in vitro on media amended with the fungicides. Following fenetrazole or fenethanil treatment, at the site where control had failed, populations of B. cinerea were detected with higher EC₅₀ and EC₉₀ values than at the three other sites. Germination of conidia of B. cinerea was markedly inhibited by 1.0 μg/ml fenetrazole and 0.5–1.0 μg/ml fenethanil. The frequency of isolates insensitive to 1.0 μg/ml fenetrazole or to 0.5 μg/ml fenethanil was 3.4 and 1.8 times higher, respectively, at the site where control had failed, compared with another site where SBI fungicides had never been applied to control grey mould. Grey mould caused by selected isolates of B. cinerea which expressed the phenotype of low sensitivity to SBI fungicides in leaves of tomato, pepper and Senecio cineraria was not controlled by either fenetrazole or fenethanil (1 .5–3.0 μg/ml). However, up to 100% disease reduction was obtained when leaves infected by sensitive isolates were treated with the fungicides.