Mycosynthesis of Silver Nanoparticles Using Screened Trichoderma Isolates and Their Antifungal Activity against Sclerotinia sclerotiorum

To control the disease caused by Sclerotinia sclerotiorum, a total of 15 isolates of the Trichoderma species was screened for the biosynthesis of silver nanoparticles (AgNPs). Among them, the highest yield occurred in the synthesis of AgNPs using a cell-free aqueous filtrate of T.virens HZA14 producing gliotoxin. The synthetic AgNPs were charactered by SEM, EDS, TEM, XRD, and FTIR. Electron microscopy studies revealed that the size of AgNPs ranged from 5–50 nm and had spherical and oval shapes with smooth surfaces. Prepared AgNPs interacted with protein, carbohydrate and heterocyclic compound molecules, and especially, interaction patterns of AgNPs with the gliotoxin molecule were proposed. The antifungal activity assays demonstrated that percentage inhibition of the prepared AgNPs was 100, 93.8 and 100% against hyphal growth, sclerotial formation, and myceliogenic germination of sclerotia at a concentration of 200 μg/mL, respectively. The direct interaction between nanoparticles and fungal cells, including AgNPs’ contact, accumulation, lamellar fragment production and micropore or fissure formation on fungal cell walls, was revealed by SEM and EDS. These will extend our understanding of the mechanisms of AgNPs’ action for preventing diversified fungal disease.

CONTROL OF WHITE MOLD DISEASE ON EGGPLANT CAUSED BY FUNGUS SCLEROTINIA SCLEROTIORUM BY INTEGRATION BETWEEN SOIL SOLARIZATION AND BIOLOGICAL CONTROL WITH FUNGUS TRICHODERMA HARZIANUM

This study was conducted in determine the efficiency of each of temperature, exposure time and bio-control agent Trichoderma harzianum in inhibition of the sclerotia myceliogenic germination of S. sclerotiorum and its mycelial growth on PDA and field. The laboratory experiment included determine efficient temperatures and its exposure hours needed to inhibit pathogen germination in order to use for minimizing disease dissemination in the field through soil solarization technique. The study showed that temperature of 60 ºC with exposure time of 5 and 6 hours were the most efficient treatments in inhibition of myceliogenic germination (55.55, 100 %) respectively. The result antagonism between T. harzianum and S. sclerotiorum by dual culture technique showed high efficacy of the bio-control agent T. harzianum inhibition anti mycelial growth of the pathogen S. sclerotiorum on PDA scoring antagonism rate of 2 degree. In terms of disease incidence the results showed that soil solarization alone and its integration with bio-control agent were completely prevented the occurrence of the disease achieving the maximum reduction in incidence rate (100 %) for all studied depths at the end of eighth weeks compared with control treatment in which the average of incidence was (85.42 %) , followed by the treatment of bio-control agent alone by a reduction rate of (42.08 %) respectively with a significant difference compared with inoculated control treatment.

TaqMan Multiplex Real-Time qPCR assays for the detection and quantification of Barley yellow dwarf virus, Wheat dwarf virus and Wheat streak mosaic virus and the study of their interactions

The phytopathogenic fungus Sclerotinia sclerotiorum forms dormant structures (termed sclerotia) that germinate myceliogenically under certain environmental conditions. During myceliogenic germination, sclerotia produce hyphae, which can infect leaves or stems of host plants directly from the ground; this is termed basal infection. This study determined which abiotic conditions were most important for promoting myceliogenic germination of sclerotia in vitro. A high sclerotium hydration level and low incubation temperature (15°C) improved mycelial growth in the presence of a nutrient source. Sclerotia incubated without a nutrient source on moist sand, vigorously myceliogenically germinated most frequently (63%) when they had been previously imbibed and then conditioned at −20°C. By far the most consistent amount of vigorous myceliogenic germination (>75%) was produced when sclerotia were heat-dried before being submerged in water. The hyphae of these sclerotia were shown to infect and proliferate on leaves of intact Brassica napus plants. This research provides a better understanding of the abiotic conditions that are likely to increase the risk of basal infection by S. sclerotiorum.

Heat-dried sclerotia of Sclerotinia sclerotiorum myceliogenically germinate in water and are able to infect Brassica napus

The phytopathogenic fungus Sclerotinia sclerotiorum forms dormant structures (termed sclerotia) that germinate myceliogenically under certain environmental conditions. During myceliogenic germination, sclerotia produce hyphae, which can infect leaves or stems of host plants directly from the ground; this is termed basal infection. This study determined which abiotic conditions were most important for promoting myceliogenic germination of sclerotia in vitro. A high sclerotium hydration level and low incubation temperature (15°C) improved mycelial growth in the presence of a nutrient source. Sclerotia incubated without a nutrient source on moist sand, vigorously myceliogenically germinated most frequently (63%) when they had been previously imbibed and then conditioned at −20°C. By far the most consistent amount of vigorous myceliogenic germination (>75%) was produced when sclerotia were heat-dried before being submerged in water. The hyphae of these sclerotia were shown to infect and proliferate on leaves of intact Brassica napus plants. This research provides a better understanding of the abiotic conditions that are likely to increase the risk of basal infection by S. sclerotiorum.

The importance of the type and time of inoculation and assessment in the determination of resistance in Brassia napus and B. juncea to Sclerotinia sclerotiorum

Sclerotinia stem rot (SSR) is a significant agricultural problem worldwide. Finding sources of resistance is crucial to the ongoing search for better management of this disease. Brassica germplasm from Australia, China and India was screened for resistance to SSR under Western Australian field conditions following stem inoculation, application of a spray of mycelial suspension, or as a consequence of myceliogenic germination originating from sclerotia resident in soil. Significant differences in response were observed among 53 genotypes using each of the three screening methods. There was a variable impact of the time of inoculation on the disease level depending upon time of assessment post-stem inoculation. However, this impact could be reduced to an insignificant level provided the assessment after stem inoculation was delayed until 3 weeks post-inoculation. The results of these studies indicate that the use of appropriate inoculation and assessment methods could significantly reduce variability in the responses commonly observed in screening for resistance in crop plants against Sclerotinia sclerotiorum.

Germination of Sclerotinia minor and S. sclerotiorum Sclerotia Under Various Soil Moisture and Temperature Combinations

Sclerotial germination of three isolates each of Sclerotinia minor and S. sclerotiorum was compared under various soil moisture and temperature combinations in soils from Huron and Salinas, CA. Sclerotia from each isolate in soil disks equilibrated at 0, -0.03, -0.07, -0.1, -0.15, and -0.3 MPa were transferred into petri plates and incubated at 5, 10, 15, 20, 25, and 30°C. Types and levels of germination in the two species were recorded. Petri plates in which apothecia were observed were transferred into a growth chamber at 15°C with a 12-h light-dark regime. All retrievable sclerotia were recovered 3 months later and tested for viability. Soil type did not affect either the type or level of germination of sclerotia. Mycelial germination was the predominant mode in sclerotia of S. minor, and it occurred between -0.03 and -0.3 MPa and 5 and 25°C, with an optimum at -0.1 MPa and 15°C. No germination occurred at 30°C or 0 MPa. Soil temperature, moisture, or soil type did not affect the viability of sclerotia of either species. Carpogenic germination of S. sclerotiorum sclerotia, measured as the number of sclerotia producing stipes and apothecia, was the predominant mode that was affected significantly by soil moisture and temperature. Myceliogenic germination in this species under the experimental conditions was infrequent. The optimum conditions for carpogenic germination were 15°C and -0.03 or -0.07 MPa. To study the effect of sclerotial size on carpogenic germination in both S. minor and S. sclerotiorum, sclerotia of three distinct size classes for each species were placed in soil disks equilibrated at -0.03 MPa and incubated at 15°C. After 6 weeks, number of stipes and apothecia produced by sclerotia were counted. Solitary S. minor sclerotia did not form apothecia, but aggregates of attached sclerotia readily formed apothecia. The number of stipes produced by both S. minor and S. sclerotiorum was highly correlated with sclerotial size. These results suggest there is a threshold of sclerotial size below which apothecia are not produced, and explains, in part, why production of apothecia in S. minor seldom occurs in nature.

Combined effects of Brassica napus seed meal and Trichoderma harzianum on two soilborne plant pathogens

The effects of soil amendment with rapeseed meal from Brassica napus cv. 'Dwarf Essex' (high glucosinolate concentrations) and 'Stonewall' (low glucosinolate concentrations) on the biological control activity of Trichoderma harzianum towards Sclerotinia sclerotiorum and Aphanomyces euteiches were evaluated. Trichoderma harzianum added to soil reduced myceliogenic germination of S. sclerotiorum by 94%, but did not affect carpogenic germination. In contrast, 100% reduction in carpogenic germination was observed in soil amended with Dwarf Essex meal, along with a 33% reduction in myceliogenic germination. With Stonewall meal as soil amendment, carpogenic germination was reduced by 44% and myceliogenic germination was not affected. Both Dwarf Essex and Stonewall meals inhibited colonization of sclerotia in soil by T. harzianum, from 100% to 0% and 8%, respectively, so that biocontrol activity of T. harzianum was reduced in the presence of either meal. Aphanomyces euteiches root rot of pea was significantly reduced by T. harzianum alone (100%), by amendment with Dwarf Essex meal alone (77%), and by T. harzianum in combination with Dwarf Essex meal (100%). Amendment with Stonewall meal alone did not control root rot, and combination of Stonewall meal with T. harzianum reduced the biocontrol efficacy of T. harzianum.

Effect of temperature during sclerotial formation, sclerotial dryness, and relative humidity on myceliogenic germination of sclerotia of Sclerotinia sclerotiorum

A study was conducted to determine the effect of sclerotial dryness, temperature during sclerotia formation, and relative humiditiy during incubation on myceliogenic germination of sclerotia of two isolates of Sclerotinia sclerotiorum (Lib.) De Bary. In the absence of exogenous nutrients, sclerotia germinated more readily at 100% RH than at 95% RH or lower. Desiccation of sclerotia is an important factor affecting myceliogenic germination and hyphal growth. At high humidity, either in an atmosphere with 100% RH or on moist sand, desiccant-dried sclerotia germinated readily and produced vigorous hyphal growth that often developed into colonies. On the other hand, fresh, untreated sclerotia germinated less readily and produced limited growth of hyphae that rarely developed into colonies. There was generally no effect of temperature at which sclerotia formed on germination. The incidence of seed rot and seedling wilt of sunflower was significantly (p < 0.05) higher when desiccant-dried sclerotia were used as inoculum rather than fresh sclerotia.Key words: Sclerotinia sclerotionum, sclerotia, myceliogenic germination, sclerotinia wilt of sunflower, relative humidity.