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.     

Virus-induced changes in the response of faba bean to infection by Botrytis

Prior infection of faba bean with the viruses bean yellow mosaic and bean leaf roll increased host susceptibility to subsequent infection by Botrytis fabae and B. cinerea. Cell necrosis beneath inoculum droplets, rate and extent of lesion spread and sporulation of B. fabae were all increased on detached leaves from virus-infected compared with healthy plants. Changes were most marked in young leaves showing conspicuous symptoms of systemic virus infection and in plants virus-infected for at least 2-4 weeks. Localized lesions produced by B. cinerea or a low concentration of B. fabae conidia (10³ spores/ ml) showed increased cell necrosis but were not transformed into aggressive, spreading lesions on virus-infected leaves.     

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.     

A rapid laboratory method for assessing the biological control potential of Penicillium oxalicum against Fusarium wilt of tomato

A rapid, simple and reliable procedure was developed to evaluate biological control of Fusarium wilt of tomato by Penicillium oxalicum . The method consists in growing tomato plants in flasks with nutrient solution in a growth chamber. Plants were previously treated in the seedbed with a conidial suspension (10⁷ conidia mL⁻¹) of P. oxalicum 7 days before transplanting. Fusarium oxysporum f.sp. lycopersici (race 2) was added to the Hoagland solution just before transplanting. Different concentrations and several isolates of F. oxysporum f.sp. lycopersici were tested. Using this method, plants showed typical symptoms of the disease and the effect of the biocontrol agent was clear. Consumption of nutrient solution was reduced in diseased plants, and this reduction was diminished by treatment with P.oxalicum . Consumption of nutrient solution was correlated with other disease-related parameters (AUDPC, weight of aerial parts, stunting) and was an easy and objective parameter to measure.     

Biocontrol of seedborne Botrytis cinerea in chickpea with Gliocladium roseum

An isolate of Gliocladium roseum proved highly antagonistic to Botrytis cinerea . Sporulation of B. cinerea on chickpea seed naturally infected or inoculated with B. cinerea was suppressed by seed treatment with conidial suspensions of G. roseum at 10⁷ and 10⁸ conidia/mL, respectively. Establishment of healthy seedlings in punnets (small trays) 5 weeks after sowing with inoculated seed was increased from 29.2% to 59.7% by treatment with G. roseum at 3×10⁷ conidia/mL, and from 1.4% to 69.4% with G. roseum at 3×10⁸ conidia/mL, the latter being equivalent to disease control by Thiram. There was no significant effect of Rhizobium on disease suppression by G. roseum , and treatment with G. roseum at 10⁸ conidia/mL did not reduce nodulation. Amendment with culture filtrates of G. roseum did not affect the growth rate of B. cinerea on potato dextrose agar, indicating that constitutive production of an antibiotic is not involved in biocontrol. A selective medium was developed to enumerate propagules of G. roseum on seed recovered from soil. There was no significant change in the population of G. roseum on seed after incubation for 4 weeks in soil to which the isolate of G. roseum was indigenous.     

Fusarium crown and root rot of tomatoes in the UK

Fusarium crown and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici was found in the UK in 1988 and 1989 mainly in rockwool-grown tomato crops. Up to 14% of plants were affected in individual crops. In experiments, leaf and stem symptoms did not appear until the time of first fruit harvest even when the plants were inoculated at planting, first flowers or fruit set. Conidial inoculum at 10⁶ spores/plant applied at seed sowing killed 70–83% of tomato seedlings, whereas similar levels of inoculum applied to young plants caused root and basal stem decay, and eventually death but only after fruit harvest began. Disease incidence and symptom severity increased with inoculum concentration. Experimentally, the disease was more severe in peat- or compost-grown plants than in rockwool. Disease spread was only a few centimetres in 50 days in experimental rockwool-grown plants. All tomato cultivars tested were highly susceptible. Prochloraz-Mn was highly effective against the pathogen in vitro and controlled the disease in the glasshouse, but only when applied preventively. Non-pathogenic Fusarium oxysporum isolates and Trichoderma harzianum also reduced FCRR disease levels.     

Factors influencing infection of eucalypts by Cylindrocladium pteridis

The pattern of Cylindrocladium pteridis adhesion, germination and penetration in eucalypt leaves was assessed using scanning electron microscopy. The effects of inoculum concentration, leaf wetness period, plant age and branch position of cylindrocladium leaf blight and defoliation severity were assessed in greenhouse studies using two Eucalyptus grandis × E. urophylla hybrid clones. Penetration occurred through stomata, and there was no difference in the number of penetrations between young and old leaves. Percentage leaf area with lesions and defoliation increased with the increase in inoculum concentration (1 × 10² to 10⁵ conidia mL⁻¹), duration of leaf wetness period (6 to 48 h) and plant age (60 to 180 days). Branch position in plants also significantly affected the percentage leaf area with lesions and defoliation, the latter variable being significantly higher at the stem base. The highest values of lesion area were also observed on leaves at the stem base in both clones. The Pearson correlation between defoliation and leaf area with lesions was significant in all experiments ( r  > 0·9) indicating a high association between these two variables.     

Visual and PCR assessment of light leaf spot (Pyrenopeziza brassicae) on winter oilseed rape (Brassica napus) cultivars

Methods to assess light leaf spot ( Pyrenopeziza brassicae ) on winter oilseed rape cultivars were compared in laboratory, controlled-environment and field experiments. In controlled-environment experiments with seedling leaves inoculated at GS 1,4, the greatest differences in percentage area affected by P. brassicae sporulation were observed with inoculum concentrations of 4 × 10³ or 4 × 10⁴ spores mL⁻¹, rather than 4 × 10² or 4 × 10⁵ spores mL⁻¹, but older leaves had begun to senesce before assessment, particularly where they were severely affected by P. brassicae . In winter oilseed rape field experiments, a severe light leaf spot epidemic developed in 2002/03 (inoculated, September/October rainfall 127·2 mm) but not in 2003/04 (uninoculated, September/October rainfall 40·7 mm). In-plot assessments discriminated between cultivars best in February/March in 2003 and June in 2004, but sometimes failed to detect plots with many infected plants (e.g. March/April 2004). Ranking of cultivar resistance differed between seedling experiments done under controlled-environment conditions and field experiments. The sensitivity of detection of P. brassicae DNA extracted from culture was greater using the PCR primer pair PbITSF/PbITSR than using primers Pb1/Pb2. P. brassicae was detected by PCR (PbITS primers) in leaves from controlled-environment experiments immediately and up to 14 days after inoculation, and in leaves sampled from field experiments 2 months before detection by visual assessment.     

Berry infection and the development of bunch rot in grapes caused by Aspergillus carbonarius

The effects of different levels of inoculum of Aspergillus carbonarius and time of inoculation on berry infection and the development of aspergillus bunch rot on grapevines (cv. Sultana) were studied under field conditions. Inflorescences at full bloom were inoculated with aqueous spore suspensions of A. carbonarius containing 0 or 1 × 10⁶ spores mL⁻¹ in 2004/05 and 0, 1 × 10² or 1 × 10⁵ spores mL⁻¹ in 2005/06. In both years, the incidence of infection in inoculated berries was significantly higher than in uninoculated berries. Incidence of infection in berries from veraison until harvest was higher than at earlier stages of bunch development (berry set to berries that were still hard and green). Inoculation of bunches at veraison did not significantly increase A. carbonarius infection prior to harvest, at harvest, 6 days after harvest or when berries were over-ripe. Bunches inoculated at harvest did not significantly increase infection 6 days after harvest or when berries were over-ripe. Aspergillus carbonarius was isolated more frequently from the pedicel end (53·1%) than from the middle section (37·5%) and distal end (35·0%) of berries that were inoculated with 10⁵ spores mL⁻¹.     

Production of conidia by Peronosclerospora sorghi on sorghum crops in Zimbabwe

Factors affecting the production of conidia of Peronosclerospora sorghi , causing sorghum downy mildew (SDM), were investigated during 1993 and 1994 in Zimbabwe. In the field conidia were detected on nights when the minimum temperature was in the range 10–19°C. On 73% of nights when conidia were detected rain had fallen within the previous 72 h and on 64% of nights wind speed was < 2.0 m s⁻¹. The time period over which conidia were detected was 2–9 h. Using incubated leaf material, conidia were produced in the temperature range 10–26°C. Local lesions and systemically infected leaf material produced 2.4–5.7 × 10³ conidia per cm². Under controlled conditions conidia were released from conidiophores for 2.5 h after maturation and were shown to be well adapted to wind dispersal, having a settling velocity of 1.5 × 10⁻⁴ m s⁻¹. Conditions that are suitable for conidia production occur in Zimbabwe and other semi-arid regions of southern Africa during the cropping season.     

Effects of wind, relative humidity, leaf movement and colony age on dispersal of conidia of Uncinula necator, causal agent of grape powdery mildew

A wind tunnel was designed to study the effect of wind, relative humidity, leaf movement and colony age on dispersal of conidia of Uncinula necator . Wind speed as low as 2.3 m s⁻¹ instantaneously triggered dispersal of conidia from fixed leaf discs of 18-day-old infections. Conidia were observed on sporulating leaf discs even after exposure to 17 m s⁻¹ wind. The fraction of conidia dispersed at a given wind speed increased with colony age from 12 to 24 days. Conidia of 27-day-old colonies were less easily dispersed. No gradient of maturation of conidia along the conidial chain was observed, suggesting that even newly formed conidia were able to germinate after dispersal. Germination of dispersed conidia decreased slightly with greater colony age. Both wind and simulated rain drops caused dispersal of conidia from infected leaves. Leaf movement at wind speed of 3.5–4 m s⁻¹ increased dispersal, and the first impact of three simulated raindrops caused release of 53% of the total conidia dispersed. Relative humidity had no effect on dispersal of conidia at different wind speeds.     

A simplified slice method for assessing tuber susceptibility of potato cultivars to Erwinia carotovora subsp atroseptica

The susceptibility of 24 potato cultivars to soft rot caused by Erwinia carotovora subsp. atroseptica was assessed in a simplified form of a tuber slice test described previously. Freshly made wounds on tuber slices were inoculated with drops (0–02 ml) containing 10⁸, 10⁷ or 10⁶ cells/ml and incubated aerobically for 3 days at 15° or 20°C. At 20°C differences between cultivars were clear at all concentrations but at 15°C less so at the lowest concentration unless incubation time was extended. The rank order of cultivar susceptibility was similar to that found in previous seasons.     

The role of phytoalexins in resistance to fusarium wilt in pigeon pea (Cajanus cajan)

Mechanisms of resistance to fusarium wilt (Fusarium udum) were investigated in pigeon pea cultivars from Malawi. Wilt-susceptible (Malawi local) and wilt resistant (ICP 9145) plants were stem-inoculated with a spore suspension containing 2·10⁶ conidia/ml of the pathogen. Occlusion of a small proportion of infected vessels was observed, but the resistant reaction appeared to depend mainly on rapid phytoalexin synthesis. Four fungitoxic isoflavonoid phytoalexins—hydroxygenistein, genistein, cajanin and cajanol-were isolated from plants 15 days after inoculation. Cajanol was identified as the main antifungal compound. The concentration of cajanol was 329·4 μg/g in the resistant cultivar as against 88·6 μg/g in the susceptible cultivar 15 days after inoculation. Crude extract from the resistant plants sampled at 24 h after inoculation contained 34·8 μg ml of cajanol. The LD₅₀ value of cajanol for spore germination was determined as 35μg/ml. The cajanol content of fungus-infected ICP 9145 10 days after inoculation totally inhibited conidial germination of F. udum.     

Effect of temperature of production of Botrytis allii conidia on their pathogenicity to harvested white onion bulbs

Botrytis allii colonies incubated at low temperatures have been reported to produce larger conidia that germinate faster and give rise to longer germ-tubes than those grown at room temperature. The present study compared the effect of conidia produced at 20°C and at 0 and –2°C on their pathogenicity to artificially inoculated white onion bulbs, and the effect of conidial concentration (5×10³ and 5×10⁴ conidia/mL) on disease incidence, lesion area, incubation and latent period during storage at 20, 5 and 0°C. At all storage temperatures and periods tested conidia produced at −2°C caused a higher disease incidence and larger areas of rot than those produced at higher temperatures. When the conidial production temperature was raised to 20°C, the duration of incubation on the bulbs inoculated with 5×10⁴ conidia/mL was more than doubled during storage at 0°C, tripled at 5°C, and took 50% longer at 20°C. The incubation period was not significantly affected by conidial concentration at 20°C, and only slightly at 5 and 0°C, but at low temperatures the latent period was longer because of the delay induced in sporulation. These data are consistent with the packers' opinion that cross-infection of spring onions by long-term refrigerated onions in grading lines caused earlier and heavier rotting.     

Cultivar dependence of polyacrylic acid effects on Pseudomonas syringae in Nicotiana tabacum

Polyacrylic acid (PAA) protected Xanthi-nc tobaccos against necrosis provoked by Pseudomonas syringae (hypersensitive reaction). When highly concentrated bacterial suspensions (1 × 10⁸ b/ml) were injected into PAA-treated leaves, tissue collapse was delayed; with more dilute bacterial suspensions (3 × 10⁷ or 1 × 10⁷ b/ml), a significant reduction or a total suppression of necrosis was observed. This phenomenon occurred when the PAA treatment was applied 4 days prior to injection of the bacteria, but not when it was applied 3 h prior to injection. No such protection occurred in Samsum NN tobaccos. PAA had no toxic effect on P. syringae when added to the injected suspensions, or in vitro in culture media. The bacterial population remained constant in protected tissue, but declined sharply in necrosing tissues. These results suggest that host metabolism is involved in PAA-induced suppression of necrosis.
Induced suppression of necrosis due to bacteria shows some analogies with induced resistance to viruses. The possibility of common steps in their biochemical mechanism is discussed.     

Effects of bean rust (Uromyces appendiculatus) epidemics on host dynamics of common bean (Phaseolus vulgaris)

Epidemics of bean rust ( Uromyces appendiculatus ) and their effects on host dynamics of common bean ( Phaseolus vulgaris ) were studied in three controlled greenhouse experiments, with and without fungicide sprays, on two susceptible bean cultivars, Dufrix and Duplika. Bean plants were artificially inoculated with a suspension of 10⁵ U. appendiculatus urediniospores mL⁻¹ water and temporal disease progress, as well as host growth dynamics (leaf size and defoliation), were monitored on a leaflet basis in comparison with non-inoculated plants, which were sprayed with deionized water. Progress curves of bean rust, expressed as the proportion of leaf area occupied by pustules (uredinia), or as the proportion occupied by total lesion area (= halo areas + pustule area), were well described by logistic functions with maximum disease levels clearly lower than 1. Bean rust epidemics substantially affected host growth by reducing the total leaf area formed by 17·4–35·6% and 35·3–46·2% compared with healthy plants for cvs Dufrix and Duplika, respectively. Fungicide sprays mitigated the negative effect of bean rust, leading to a gain in leaf area of 17–21% compared with unsprayed plants in both cultivars in two experiments, while in another experiment, disease control had no effect in Dufrix, but a clear effect in Duplika. In addition to the growth depression, bean rust also led to pronounced losses of leaf area as a result of reduced leaf size (leaf shrivelling) and accelerated defoliation.     

Effects of benzothiadiazole and acetylsalicylic acid on β-1,3-glucanase activity and disease resistance in potato

Benzothiadiazole (BTH), as Bion WG50, and acetylsalicylic acid (ASA) treatments of potato foliage of field- and glasshouse-grown potato plants were compared for control of two foliar diseases, early blight ( Alternaria solani ) and powdery mildew ( Erysiphe cichoracearum ). The effect of these treatments on harvested tubers wound-inoculated with the dry rot fungus ( Fusarium semitectum ) was also evaluated. BTH (50 mg a.i. L⁻¹) gave almost complete control of both foliar pathogens on inoculated glasshouse-grown plants and reduced the severity of leaf spotting diseases (mainly early blight) in the field. BTH (100 mg a.i. L⁻¹) and ASA (400 mg a.i. L⁻¹) reduced the severity of dry rot in field-grown tubers in some post-harvest wound-inoculated treatments but not others and a similar reduction occurred with tubers inoculated post-harvest from BTH-treated plants grown under glasshouse conditions. BTH treatment increased β-1,3-glucanase activity in leaves > stem > tubers > stolons but not in roots. Increased enzyme activity was recorded for up to 45 days post-treatment.     

Factors influencing infection of mechanical wounds by Fusarium circinatum on Monterey pines (Pinus radiata)

Pitch canker, caused by the fungus Fusarium circinatum , is a disease affecting many pine tree species. In California, Pinus radiata (Monterey pine) is the principal pine host affected by pitch canker. This investigation into factors affecting infection frequency by F. circinatum of P. radiata examined the influence of: (i) wound size; (ii) relative humidity; (iii) time of inoculation; (iv) inoculum density; and (v) wound age. Wounded branches sustained significantly more infections when large-diameter (1·6 mm) rather than small-diameter (0·5 mm) wounds were made. Infection frequencies tended to be higher at 100% RH than at ambient humidity, although these differences were not statistically significant. Infection frequencies were significantly higher on branches inoculated after 17·00 h than on branches inoculated before noon. Infection frequencies were significantly higher for wounded branches spray-inoculated with 5 × 10⁷ rather than 1 × 10⁷ spores mL⁻¹. Infection frequencies of pruning wounds declined as wounds aged.     

Effects of the safeners CGA 154281, oxabetrinil and fenclorim on uptake and degradation of metolachlor in corn (Zea mays L.) seedlings

Summary. The influence of three crop safeners on the uptake and degradation of ¹⁴C-metolachlor was investigated in two corn varieties. Following application of herbicide and safener together to seedling shoots the concentrations of non-metabolized ¹⁴C-metolachlor in the tissues was found to be lower in the tolerant variety LG 9 than in the susceptible variety 211A. The difference between varieties was due to differences in both uptake and degradation of ¹⁴C-metolachlor.
Following shoot application most of the radioactivity was retained in the coleoptile and the mesocotyl. Two hours after application 95% of the herbicide had been degraded in coleoptiles and mesocotyls, whereas approximately 20% of non-metabolized ¹⁴C-metolachlor was present in the enclosed developing shoot leaves. In both corn varieties the safener CGA 154281 caused a substantial lowering of tissue levels of parent ¹⁴C-metolachlor. This was primarily due to an enhanced degradation. Glutalhione- S -transfer-ase (GST) enzyme activity in shoot tissues was found to be enhanced in both varieties by CGA 154281. Oxabetrinil and fenclorim were less effective than CGA 154281 both in reducing tissue levels of non-metabolized ¹⁴C-metolachlor and in enhancing GST activity in either variety.     

Contamination and subsequent multiplication of soft rot erwinias on healthy potato leaves and debris after haulm destruction

Small plots of potatoes were inoculated with a mixture of Erwinia carotovora (E. c.) subsp. carotovora and E. carotovora subsp, atroseptica strains resistant to rifampicin. Subsequently the population off, c. subsp, carotovora and E. c. subsp, atroseptica (rifampicin-resistant and wild types) present as epiphytes on the surface of potato leaves was assessed using three methods, qualitative, semi-quantitative and quantitative, during 1986 and 1987. The population was generally low (< 10² colony forming units (> 10⁴cfu/g leaves) but reached higher levels (> 10⁴ cfu/g) on occasions later in the growing season, Rifampicin-resistant erwinias were reisolated only infrequently throughout this study. Different methods of haulm destruction (herbicide, pulverization, sulphuric acid treatment and natural senescence) greatly influenced the number of erwinias present in the resulting plant debris. Pulverization resulted in the highest population (10⁶-10⁷ wild-type cfu/g) in both seasons. In 1987. the wettest of the two seasons of this study, herbicide treatment resulted in similarly high populations. The results suggest that the high numbers of erwinias found in the haulm debris were probably derived from the generally low populations of epiphytic bacteria previously present on healthy leaves, E. c. subsp, carotovora was the most frequent subspecies in the rotting plant debris; E. c. subsp, atroseptica was more commonly found on healthy leaves. The implications of the results are discussed in relation to the production of seed potatoes with a low risk of blackleg.