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 the mycoparasite Verticillium biguttatum on barley stunt disease caused by Rhizoctonia solani anastomosis group 8 in a model system

The height of barley stunted by Rhizoctonia solani anastomosis group 8 was significantly increased by up to 72·8% after incubation for 8 days at 20°C in seedling tray tests following application of the mycoparasite Verticillium biguttatum. The pathogen and mycoparasite were applied at the rate of 1% Perlite maizemeal inoculum (w/w potting mixture) resulting in propagule densities of approximately 24·0 and 6·6 × 10⁵ colony-forming units (cfu) per g potting mixture, respectively. Sieving (2 mm) R. solani inoculum prior to dilution in potting mixture increased the recovery of propagules from 1·2 × 2·1 × 10³ cfu per g inoculum compared with recovery when inoculum was sieved after dilution. Applications of a V. biguttatum isolate from the UK (vbl) and a Dutch isolate (M73) reduced stunting to a similar extent but did not stimulate the growth of healthy plants. The height of stunted plants was significantly increased after application of V. biguttatum inoculum after 6 days if inoculated trays were preincubated for 1 day prior to planting but a similar increase was only detected after 7 days if seeds were planted immediately. The number of stunted plants which emerged after 4 days was significantly increased by treatment with V. biguttatum but preincubation had no additional effect. These results suggested that control of R. solani was effected both before and after the initiation of disease.     

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.     

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.     

Sporulation of Pyrenopeziza brassicae (light leaf spot) on oilseed rape (Brassica napus) leaves inoculated with ascospores or conidia at different temperatures and wetness durations

In controlled environment experiments, sporulation of Pyrenopeziza brassicae was observed on leaves of oilseed rape inoculated with ascospores or conidia at temperatures from 8 to 20°C at all leaf wetness durations from 6 to 72 h, except after 6 h leaf wetness duration at 8°C. The shortest times from inoculation to first observed sporulation ( l ₀), for both ascospore and conidial inoculum, were 11–12 days at 16°C after 48 h wetness duration. For both ascospore and conidial inoculum (48 h wetness duration), the number of conidia produced per cm² leaf area with sporulation was seven to eight times less at 20°C than at 8, 12 or 16°C. Values of Gompertz parameters c (maximum percentage leaf area with sporulation), r (maximum rate of increase in percentage leaf area with sporulation) and l ₃₇ (days from inoculation to 37% of maximum sporulation), estimated by fitting the equation to the observed data, were linearly related to values predicted by inserting temperature and wetness duration treatment values into existing equations. The observed data were fitted better by logistic equations than by Gompertz equations (which overestimated at low temperatures). For both ascospore and conidial inoculum, the latent period derived from the logistic equation (days from inoculation to 50% of maximum sporulation, l ₅₀) of P. brassicae was generally shortest at 16°C, and increased as temperature increased to 20°C or decreased to 8°C. Minimum numbers of spores needed to produce sporulation on leaves were ≈25 ascospores per leaf and ≈700 conidia per leaf, at 16°C after 48 h leaf wetness duration.     

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.     

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.     

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.     

Bioassays of glucosinolate-derived isothiocyanates against postharvest pear pathogens

The present study assayed the effect of six isothiocyanates (ITCs), produced by the enzymatic hydrolysis of glucosinolates, on fungal pathogens of pear. Sample pear fruits were artificially inoculated through induced wounds with conidial suspensions of Botrytis cinerea Rhizopus stolonifer Monilinia laxa Mucor piriformis or Penicillium expansum and were then treated with ITCs. Of the six ITCs tested, the ITC from glucoraphenin showed the highest effectiveness after 6 days at 20°C, against M. laxa B. cinerea and M. piriformis . The effectiveness of the ITC from glucoraphenin against M. laxa was assayed in two further trials to test the effect of ITC concentration on different concentrations of inoculum and to determine the duration of the curative effect of this ITC. ITC concentration directly affected fungus control capacity. The highest ITC concentration (3.6 mg mL⁻¹) afforded pathogen control at the highest level of pathogen concentration (10⁶ conidia mL⁻¹) after 6 days at 20°C. Its curative effect was evident up to 40 h after inoculation.     

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.     

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.     

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.     

Epidemiological studies on cercospora leaf spot of sugar beet

In the glasshouse, inoculation of sugar beet with Cercospora beticola followed by 16 h of high humidity produced visible disease only with at least four conidia per cm² of leaf area. Disease became more severe after increasing periods of high humidity in the range of 0–24 h. In the field, spraying plants with water enhanced disease spread from a focus. Disease progress curves were sigmoid. Apparent infection rate declined towards the end of the season, possibly because of high temperature. In approximate agreement with prediction, epidemic development was delayed when initial inoculum was reduced. Reduced infection, resulting from either reduced initial inoculum or delayed inoculation, decreased the adverse effect of disease on sugar yield.     

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.     

The use of a tomato cotyledon test to identify Corynebacterium michiganense pv. michiganense

When cotyledons of 4-day-old tomato seedlings (Lycopersicon esculentum cv. Sonato) were inoculated with Corynebacterium michiganense pv. michiganense and kept at 25°C with a 16 h light period per day, white raised blisters were produced which coalesced to give white, scurfy, cracked areas after 3 days. This symptom did not develop when Gram-positive coryneform bacteria isolated from, and near, tomato crops were used as inoculum. Pre-inoculation abrasion of cotyledons, an inoculum concentration of at least 10⁵ bacteria/ml, and high humidity after inoculation were important factors in the expression of this host reaction. The cotyledon test may be of value in laboratories where more detailed tests for C. michiganense pv. michiganense are not possible.     

Biological control of southern blight disease of tomato caused by Sclerotium rolfsii with simplified mycelial formulations of Trichoderma koningii

Two simple formulations of an antagonistic strain of Trichoderma koningii were employed against southern blight disease caused by Sclerotium rolfsii in seedling, potted outdoor and field-grown tomatoes (cvs. Ife No. 1 and Ibadan Local). Corn cob germling inoculum and mycelium powder of T. koningii significantly controlled ( P ≤0·05) symptoms of damping off, blight and wilting in both tomato cultivars. The populations of the antagonist increased from an initial 1 × 10⁴ to about 1 × 10⁶ colony-forming units per g of soil in the protected plants. Moreover, sclerotial counts decreased significantly ( P ≤0·05) in these soils and those sclerotia found had been parasitized by T. koningii. Trichoderma -protected plants were more vigorous than those in the other treatment categories. The significance of these results is discussed in relation to the use of Trichoderma in appropriately simplified formulations.     

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.     

Factors affecting the incidence and severity of Spongospora subterranea infection and galling in potato roots

The incidence and severity of root infection and root galling caused by Spongospora subterranea were assessed in potato plants (cv. Estima) grown under controlled environmental conditions. The effects of temperature, soil type, soil moisture regime and soil inoculum level on infection and root gall development were determined by molecular and visual methods at two plant growth stages. Root gall severity was scored at harvest, after which DNA was extracted from the roots and quantified in a real-time polymerase chain reaction (PCR) assay specific for S. subterranea . Root galling was severe at 17°C, with a disease score of 3·1 on a 0–4 scale, low (0·6) at 12°C, and did not occur at 9°C. The level of inoculum in soil, in the form of artificially added sporosori, had no effect on the incidence and severity of visual symptoms, with 21%, 41% and 33% incidence observed at 5, 15 and 50 sporosori g⁻¹ soil, respectively. Incidence of infection, as detected by the real-time PCR assay, was greater with increasing soil inoculum concentrations, ranging from 48% at 5 sporosori g⁻¹ to 59% (15 sporosori g⁻¹) and 73% (50 sporosori g⁻¹) of plants infected at maturity, but this effect was not statistically significant. No correlation was found between the occurrence of galls on roots and powdery scab on tubers of the same plants.     

Use of Trichoderma harzianum in combination or alternation with fungicides to control cucumber grey mould (Botrytis cinerea) under commercial greenhouse conditions

A preparation of Trichoderma harzianum was sprayed on cucumber plants in greenhouses in order to control fruit and stem grey mould. Up to 90% control was achieved by the biocontrol agent (0·5–1·0 g/l) which in most experiments under commercial conditions was as effective as the dicarboximide fungicides iprodione or vinclozolin (0·5 g/l each) alone or alternated with diethofencarb + carbendazim (0·25 g/l each). However, in one experiment disease incidence in Trichoderma -treated plots did not differ significantly from the control. A mixture of T. harzianum with a dicarboximide fungicide resulted in up to 96% control of grey mould. In this case control was always significant ( P =0·05) but improvement of control compared with each treatment alone was not significant ( P =0·05). The alternation of sprays with the biocontrol preparation and with a dicarboximide fungicide was tested in three out of the five experiments and was found to be effective, thus enabling a reduction in the use of chemical sprays. Populations of T. harzianum were on a level of 3 × 10⁵-8 × 10⁵ c.f.u. per leaf and ten times lower on one fruit. They remained high after the second and third sprays. Conditions favouring the ability of T. harzianum to control grey mould were temperatures above 20°C and relative humidity between 80 and 97%.     

Effects of nitrogen, phosphorus, potassium and calcium nutrition on strawberry anthracnose

The effects of a range of concentrations of four nutrients – nitrogen, phosphorus, potassium and calcium – in fertilizer solutions on the severity of anthracnose on strawberry cv. Nyoho cultivated under a noncirculation hydroponics system were determined after inoculation with Colletotrichum gloeosporioides . Crop growth and tissue nitrogen, phosphorus, potassium and calcium contents of the entire above-ground parts of the plant were also investigated. Elevated nitrogen and potassium concentrations in the fertilizer solution increased disease severity in contrast to phosphorus and calcium. Treatment with either NH₄ or NO₃ nitrogen was not significantly different. The dry weight of the strawberry plants increased significantly with elevated concentrations of nitrogen ( R ² = 0·9078) and phosphorus ( R ² = 0·8842), but was not influenced by the elevated amounts of potassium ( R ² = 0·8587) and calcium ( R ² = 0·6526) concentrations.