Environmental Effects of Temperature and Water Potential on Mycelial Growth of Neocosmospora solani and Fusarium spp. Causing Dry Root Rot of Citrus.

This study aimed at evaluating the effect of environmental factors temperature and water potential (Ψw) on the growth of Neocosmospora (Fusarium) solani and three Fusarium species (F. oxysporum, F. equiseti and F. brachygibbosum) associated with citrus dry root rot and to determine the optimum and marginal rate for their growth. The effects of incubation temperature (5-40 °C), water potentials (Ψw) (- 15.54; - 0.67 MPa) (0.89-0.995 aw) and their interaction (5-30 °C) was evaluated on the in vitro radial growth rates of Fusarium spp. and on their lag phase. Secondary models were used to model the combined effect of these factors on radial growth rate. The results underlined a highly significant effects (P < 0.001) of Ψw and temperature and their interactions on radial growth rates and lag phases (λ). The Four studied species were shown tolerant to a temperature of 35 °C with an optimum mycelial growth at 30 for N. solani and F. oxysporum and at 25 °C for F. equiseti and F. brachygibbosum. However, no growth was observed at both temperatures 5 and 40 °C and at Ψw of - 9.68 MPa (0.93 aw). The optimum water potential for growth was ≥- 2.69 MPA (>0.98 aw). The results from the polynomial model and response surface showing good agreement between observed and predicted values. The external validation on citrus fruit indicated slight differences between predicted and observed values of radial growth. The results of this study will be beneficial for understanding the ecological knowledge of these species and thereby limited preventively their occurrence.

Evidence that the biofungicide Serenade (Bacillus subtilis) suppresses clubroot on canola via antibiosis and induced host resistance.

This study investigated how the timing of application of the biofungicide Serenade (Bacillus subtilis QST713) or it components (product filtrate and bacterial cell suspension) influenced infection of canola by Plasmodiophora brassicae under controlled conditions. The biofungicide and its components were applied as a soil drench at 5% concentration (vol/vol or equivalent CFU) to a planting mix infested with P. brassicae at seeding or at transplanting 7 or 14 days after seeding (DAS) to target primary and secondary zoospores of P. brassicae. Quantitative polymerase chain reaction (qPCR) was used to assess root colonization by B. subtilis as well as P. brassicae. The biofungicide was consistently more effective than the individual components in reducing infection by P. brassicae. Two applications were more effective than one, with the biofungicide suppressing infection completely and the individual components reducing clubroot severity by 62 to 83%. The biofungicide also reduced genomic DNA of P. brassicae in canola roots by 26 to 99% at 7 and 14 DAS, and the qPCR results were strongly correlated with root hair infection (%) assessed at the same time (r = 0.84 to 0.95). qPCR was also used to quantify the transcript activity of nine host-defense-related genes in inoculated plants treated with Serenade at 14 DAS for potential induced resistance. Genes encoding the jasmonic acid (BnOPR2), ethylene (BnACO), and phenylpropanoid (BnOPCL and BnCCR) pathways were upregulated by 2.2- to 23-fold in plants treated with the biofungicide relative to control plants. This induced defense response was translocated to the foliage (determined based on the inhibition of infection by Leptosphaeria maculans). It is possible that antibiosis and induced resistance are involved in clubroot suppression by Serenade. Activity against the infection from both primary and secondary zoospores of P. brassicae may be required for maximum efficacy against clubroot.

Effect of temperature and water activity on spore germination and mycelial growth of three fungal biocontrol agents against water hyacinth (Eichhornia crassipes).

AIMS: To determine the effect of water activity (a(w) =0·880-0·960) and temperature (15-35°C) on the percentage of viable conidia and mycelial growth of three biocontrol agents effective against water hyacinth in Mali: Alternaria sp. isolate Mlb684, Fusarium sacchari isolate Mln799 and Cadophora malorum isolate Mln715. METHODS AND RESULTS: The fungi were grown in vitro on plates containing potato dextrose agar medium at different a(w) values (glycerol being added to adjust the a(w)). The percentage of viable conidia and radial growth rate decreased with decreasing water activity. Statistical analysis showed a significant effect of a(w), temperature and the a(w) × temperature interaction on mycelial growth (P<0·0001). Water activity emerged as the factor exerting the greatest influence. Differences were observed between the fungi tested, the C. malorum appearing more tolerant to low a(w) and the F. sacchari more tolerant to high temperature (35°C). Growth models predicting the combined effect of a(w) and temperature were developed and response surfaces generated, showing fairly good agreement with the experimental values. CONCLUSIONS: Our results confirm the previous finding that a(w) has a greater influence than temperature on fungal growth. Under most conditions, variation of environmental factors has a detrimental influence on the percentage of viable conidia and mycelial growth rate of fungal isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed models may contribute to predicting the best environmental conditions for use of these fungi as effective biocontrol agents against water hyacinth.

Response surface methodology study of the combined effects of temperature, pH, and aw on the growth rate of Trichoderma asperellum.

AIMS: To evaluate the influence of environmental parameters (water activity aw, temperature, and pH) on the radial growth rate of Trichoderma asperellum (strains PR10, PR11, PR12, and 659-7), an antagonist of Phytophthora megakarya, the causal agent of cocoa black pod disease. METHODS AND RESULTS: The radial growth of four strains of T. asperellum was monitored for 30 days on modified PDA medium. Six levels of aw (0.995, 0.980, 0.960, 0.930, 0.910, and 0.880) were combined with three values of pH (4.5, 6.5, and 8.5) and three incubation temperatures (20, 25, and 30 degrees C). Whatever the strain, mycelial growth rate was optimal at aw between 0.995 and 0.980, independently of the temperature and pH. Each strain appeared to be very sensitive to aw reduction. In addition, all four strains were able to grow at all temperatures and pH values (4.5-8.5) tested, highest growth rate being observed at 30 degrees C and at pH 4.5-6.5. The use of response surface methodology to model the combined effects of aw, temperature, and pH on the radial growth rate of the T. asperellum strains confirmed the observed results. In our model, growth of the T. asperellum strains showed a greater dependence on aw than on temperature or pH under in vitro conditions. CONCLUSION: aw is a crucial environmental factor. Low aw can prevent growth of T. asperellum strains under some conditions. The observed and predicted radial growth rate of strain PR11 showed its greater capacity to support low aw (0.93) as compared with other tested strains at 20 degrees C. This is in agreement with its better protective level when applied in medium-scale trials on cocoa plantations. SIGNIFICANCE AND IMPACT OF THE STUDY: This study should contribute towards improving the biocontrol efficacy of T. asperellum strains used against P. megakarya. Integrated into a broader study of the impact of environmental factors on the biocontrol agent-pathogen system, this work should help to build a more rational control strategy, possibly involving the use of a compatible adjuvant protecting T. asperellum against desiccation.

Isolation and evaluation of bacteria and fungi as biological control agents against Rhizoctonia solani.

Rhizoctonia solani is one of the most important limiting factors for potato production and storage in Belgium and worldwide. Its management is still strongly dependent on chemical treatments. The aim of this work was to evaluate the possibility of exploiting bacteria and fungi in order to control this pathogen. Among a collection of 220 bacterial strains isolated from different organs of healthy potato plants and rhizospheric soils, 25 isolates were selected using screening methods based on in vitro dual culture assays. The mycelial growth inhibition rate of the pathogen was ranged from 59.4 to 95.0%. Also seven fungal strains isolated from the rhizospheric soil and potato roots showed a highly mycelial growth inhibition of R. solani. The mycelial growth inhibition rate obtained with these fungi was included between 60.0 and 99.4%. From this preliminary study, the further investigations will be planned to determine the bacterial isolates systematic, species of fungal strains by using molecular tools and to assess their efficacy against R. solani in greenhouse trials.

In vitro effects of water activity, temperature and solutes on the growth rate of P. italicum Wehmer and P. digitatum Sacc.

AIMS: To evaluate the effect of water activity (a(w) 0.98-0.89, adjusted with glycerol, sorbitol, glucose, or NaCl) and temperature (5-25 degrees C) on the lag phase and radial growth rate (mm day(-1)) of the important citrus spoilage fungi, such as Penicillium italicum and Penicillium digitatum grown in potato dextrose agar (PDA) medium. To select, among models based on the use of different solutes, a model fitting accurately the growth of these species in relation to a(w) and temperature. METHODS AND RESULTS: Extensive data analyses showed for both Penicillium species a highly significant effect of a(w), temperature, solutes and their interactions on radial growth rate (P < 0.0001). Radial growth rate was inhibited and the lag phase (i.e. the time required for growth) lengthened as the a(w) of the medium decreased. NaCl appeared to causes the greatest stress on growth when compared with other nonionic solutes. Penicillium italicum stopped growing at 0.96 a(w) and P. digitatum at 0.93 a(w). Under the dry conditions where growth was observed, P. italicum grew faster than P. digitatum at low temperature and P. digitatum remained more active at ambient temperature. Multiple regression analysis applied to the square roots of the growth rates observed in the presence of each solute showed that both the 'glycerol model' and the 'sorbitol model' yielded a good prediction of P. italicum growth and the 'sorbitol model' gave an accurate fit for P. digitatum growth, offering high-quality prediction within the experimental limits described. CONCLUSIONS: Mathematical models describing and predicting, as a function of a(w) and temperature, the square root of the radial growth rate of the agents responsible for blue and green decays are important tools for understanding the behaviour of these fungi under natural conditions and for predicting citrus fruit spoilage. SIGNIFICANCE AND IMPACT OF THE STUDY: Implementation of these results should contribute towards a more rational control strategy against citrus spoilage fungi.

Effect of incubation temperature and relative humidity on lesion diameter of Botrytis cinerea Pers. and Penicillium expansum Link. on apple fruits.

Previous studies carried out in our laboratory demonstrated that the growth of B. cinerea and P. expansum was highly influenced by water activity and temperature in 'in vitro' conditions. Regardless the temperature, a, minimal for growth was < or = 0.89 and equal to 0.89 for P. expansum and B. cinerea respectively. The effect of incubation temperature (5-25 degrees C) and relative humidity (RH 75-98%) on the lesion diameter of two common fungi B. cinerea and P. expansum was studied and modelled in the controlled laboratory conditions in order to validate previous findings. The obtained results showed that only temperature had a significant effect on fungal growth on wounded apples. The relative humidity of air had no direct influence on growth of fungi. The part of variation explained by both studied factors is 52 and 55% respectively for P. expansum and B. cinerea. 'Lack of Fit' test was no significant for models, suggesting a greater difference between observed and predicted values. The difference between observed and predicted values was 14 and 29% respectively for P. expansum and B. cinerea. Theses results is in contradiction as compared to in vitro conditions and underlined that the humidity inside wounded apple sites is highly sufficient to start the growth of both postharvest pathogens of apples.

Studying and modelling the combined effect of temperature and water activity on the growth rate of P. expansum.

The effect of solutes, water activity (a(w), 0.890--0.980) and temperature (5--25 degrees C) on the mycelial growth rate of Penicillium expansum was evaluated. The growth rate dropped as the temperature and a(w) of the medium decreased. NaCl was the solute causing the greatest growth rate reduction, followed by glucose, glycerol and sorbitol. Statistical analysis of the results showed a significant effect of solute, a(w), temperature and combinations of two or three of these factors (P<0.0001). Whatever the solutes and a(w) values, the initiation of colony growth required an additional day at 15 degrees C and 5 degrees C as compared to initiation at 25 degrees C. Growth models based on the results obtained with sorbitol and glycerol differed only slightly, with R(2) values of 97.00% and 97.95%, respectively. The response surfaces of both quadratic polynomial models showed that P. expansum should be able to grow at low a(w) (0.890) and that growth at 25 degrees C should be fastest at a(w) values ranging from 0.960 to 0.980. Both models presented a good fit between predicted and observed values.

Development of a biological control method against postharvest diseases of citrus fruits.

Candida oleophila strain O was previously selected for its high and reliable antagonistic activity against Botrytis cinerea and Penicillium expansum, two important wound pathogens on post-harvest apples. The application of these antagonistic strains on wound pathogens of Citrus was more recently undertaken. The efficacy of yeast (applied at several concentrations from 10(5) to 10(8) CFU/ml) was assessed against P. digitatum and P. italicum inoculated after one hours (at a concentration of 10(5), 106 and 10(7) spores/ml) on 'Clementine' and 'Valencia late' varieties. The protective levels were positively correlated with high concentration of antagonist and low concentration of pathogen. The antagonistic activity of this strain was also dependent on the incubation time before pathogen inoculation. The protective level increased with time between application of the antagonist and inoculation of fungal spores. Finally, the efficacy of biomass of C. oleophila strain O (produced at an industrial scale), and two different formulations of that biomass was assessed in comparison with fungicidal treatment (Thiabendazole) under semi-practical conditions against P. digitatum. This efficacy of strain O (whatever its formulation) was statistically comparable to that for TBZ at commercial dose, indicating that both formulations could be used as an alternative for conventional fungicide in postharvest treatments.

Efficacy assessment of Candida oleophila (strain O) and Pichia anomala (strain K) against major postharvest diseases of citrus fruits in Morocco.

Two yeasts, Candida oleophila (strain O) and Pichia anomala (strain K), were previously selected for their antagonistic activity against postharvest diseases on apples and pears. The objective of the study was to determine the efficacy of both antagonistic yeast's against wound postharvest pathogens of citrus fruits. The efficacy of both strains (applied at 10(5), 10(6) and 10(8) CFU/ml) was assessed against Penicillium digitatum and P. italicum inoculated after one hour (at a concentration of 10(5), 10(6) and 10(7) spores/ml) on citrus varieties 'clementine' and 'valencia-late'. Fruits were incubated for one week at 24 degrees C before measurement of lesion diameter. The protective levels were positively correlated with high concentration of antagonist and low concentration of pathogen. Highest protective levels (from 73 to 100%) were detected with the application of strain O or strain K at 10(8) CFU/ml whatever the pathogen (applied at 10(5) spores/ml) and the citrus variety. The antagonistic activity of both strains was also dependent on the incubation period before pathogen Inoculation. The protective level increased with time between application of the antagonist and inoculation of fungal spores. Whatever the yeast strain (10(8) CFU/ml). the protective level exceed 70% when wounded oranges were inoculated with P. digitatum or P. italicum (both at 10(6) spores/ml) 12 hours after yeast treatment. These protective levels reached 100% when the incubation period separating the antagonist application and the pathogenic inoculation was 24 hours. On the other hand, high protective levels (< 80%) were also observed against the sour rot decay on citrus variety 'clementine' caused by Geotrichum candidum inoculated at concentration of 10(6) spores/ml when strain O or strain K were applied at 10(8) CFU/ml 24 hours before pathogen. All these results support the potential practical application of both strains against major postharvest pathogens on citrus.