MATERIALS AND METHODS

Laboratory studies (Isolation, purification and identification of the causal organisms)
Isolation: The collected naturally infected roots of the diseased sesame plants were thoroughly washed with running tap water for several times to remove the adhered soil particles. These roots were then cut into small pieces (0.5-1 cm), excised tissues were surface disinfected by immersing them into sodium hypochlorite solution (0.5%) for 1-1.5 min, passed in sterilized distilled water and dried between two sterilized filter papers. After that, sterilized pieces were separately placed into Petri plates (9 cm) containing Acidified Potato Dextrose Agar (APDA) medium at the rate of 4 or 5 pieces for each dish. Plates were carefully closed with Para-film before incubation at 28±2°C for two weeks¹⁴.

Purification: During the incubation period, any emerged fungus was purified using the hyphal tip technique¹⁵.

Identification: Plates were examined daily for two weeks and the developed fungi were identified according to their morphological and cultural features^16-18.

The effects of three Brassica on Macrophomina phaseolina growth in vitro: These studies were carried out at Plant Pathology Laboratory, Plant Production Department, Faculty of Environ. Agricultural Science, Al-Arish between 2017-2018. This experiment was carried out to compare the effects of volatiles released from three crops i.e. radish (Raphanus sativus), cauliflower (Brassica oleracea var. botrytis) and rocket (Eruca sativa) on Macrophomina phaseolina growth. Tested crops seeds were planted at the experimental farm of Faculty of Environmental Agriculture Science., El-Arish.

The Agar plugs (6 mm diameter) were taken from the edge of M. phaseolina colonies actively growing in APDA medium (27°C, 7 days of incubation in the dark) were cut and transferred to the center of Petri dishes (9 cm diameter) containing fresh APDA medium.

Tissues including root pieces and green above-ground tissues in equal amounts were collected at the 10-leaf stage and washed with tap water and dried out. The tissues were disinfected in ethanol 10% for 10 sec, rinsed in sterile distilled water (SDW) for 5 min, dried on autoclaved filtering paper and macerated using a sterile mortar and pestle.

Two grams of fresh macerated tissues of each of the three Brassica species were placed in the lids of each Petri dish under sterile conditions. All dishes, including plates without any plant tissues (control), were sealed carefully with two layers of parafilm to prevent any possible vapor leak of volatiles from those plates. Plates were incubated upside down at 27°C for two weeks. The diameter of M. phaseolina colonies was measured 7 and 14 days after incubation. Each treatment (plant tissue, pathogen combination) had four replicates. This experiment was repeated twice during the 2017 and 2018 seasons.

As for the tested fungicide (Rhizolex-T), (50% WP) it was added to APDA medium before solidification under aseptic conditions. The media was poured in Petri dishes (9 cm) with four replicates. Similar to the Brassica tissue treatments, agar plugs (6 mm) taken from the edge of 7 days old cultures of M. phaseolina were transferred to the center of the agar surface of every replicate. Plates were also incubated at 27°C for two weeks. All tested dishes were incubated as mentioned before. linear growth of tested dishes was measured by measuring two perpendicular diameters in cm and the average was recorded and the percent reduction of radial growth in each treatment was measured 7-14 days after inoculation by the formula Abdullah et al.¹⁹:

where, I is percent growth inhibition, C is colony diameter of the pathogen in control and T is colony diameter of the pathogen in treatment. This experiment was conducted during two seasons with four replicate for each treatment.

Field studies
Preceding crops planting: Cauliflower seeds were sown in the nursery November, 14 and 23 during the 2017 and 2018 seasons, respectively. Radish and Watercress were sown in the field directly. The ideal agricultural practices were performed until seeds and cauliflower seedlings were transplanted into the field on December, 25 and 30 during the 2017 and 2018 seasons, respectively.

Biofumigation and soil solarization treatments:

Control treatment: Control plants remained untreated and uncovered but were similarly irrigated to field capacity.

Fungicide treatment: Soil drenching with the fungicide was applied at three intervals, i.e. 15, 30 and 45 days after transplanting. The application rates were 1.5 gm/I water for the chemical fungicide Rhizolex-T 50% WP. The fungicide was applied individually. Each plant received 250 ml of the tested fungicide solution²³.

Sesame planting: Sesame seeds of Chandawel 3 cultivar were sown in soil directly on two different dates. The ideal agricultural practices were performed after previous crops were removed. The experiment consisted of 12 rows 8.5 m long and 6 m width. Each row contained 34 plants at 25 cm distances in-betweens. The experiment contained six treatments with four replicates, for each treatment. Plants were irrigated using a drip irrigation system. Fertilization was done through the drip irrigation system weekly. The ideal agricultural practices were carried out as usual²⁴.

Measurements
Disease severity index: Disease severity of root rot and any discoloration of tissue were recorded according to Haware and Nene²⁵ based on 0-4 scale according to percentage of foliage yellowing or necrosis (0 = 0%, 1 = 1-33%, 2 = 34-66%, 3 = 67-l00% and 4 = dead plant)

Statistical analysis: Data were statistically analyzed using MSTAT computer program. The least significant difference (LSD) at 0.05 level was used for comparing the differences between means.

RESULTS AND DISCUSSION

Laboratory studies
Effect of three Brassica species on mycelial growth of Macrophomina phaseolina, in vitro: This experiment was carried out to study the effects of volatile compounds released by macerated tissues of three Brassica species i.e. cauliflower, radish and rocket as compared with the fungicide Rhizolex-T on mycelial growth of Macrophomina phaseolina in vitro.

All tested Brassica species induced growth reduction of M. phaseolina (Table 1) in both experiments (2017, 2018). Cauliflower and radish were significantly more effective than a rocket in M. phaseolina growth suppression.

Table 1 showed that the effect of volatile inhibitors produced from fresh macerated tissues against M. Phaseolina isolate growth was significantly different from untreated controls. Comparing the Rhizolex-T treatment with the control treatment, it appears that the fungicide significantly reduced M. phaseolina growth by 39.5 and 38.79% in both seasons respectively. As for the Cauliflower treatment also resulted in a significant reduction in M. Phaseolina radial growth with 26.12 and 20.15% in both seasons, respectively. In addition, the rocket was the least effective treatment in suppressing the mycelial growth of M. phaseolina with 9.18 and 10. 40% reduction, respectively.

The suppression of fungal mycelial growth using various Brassica species has been reported by various researchers^26-28.

Fields studies
Effects of biofumigation on root rot of sesame
Disease severity index (DSI): Data in Table 2 revealed that biofumigation significantly reduced root rot disease of sesame as compared with the control treatment. Cauliflower-biofumigation and Rhizolex-T-T showed the lowest level of disease severity index of (2.000, 2.000) at (80 days after transplanting) in 2017, respectively.

Table 1:	Effects of volatiles released from three species of Brassica on linear growth (cm) of Macrophomina phaseolina
*Means in column followed by the same alphabetical letter are not significantly different at 5% level according to LSD, *Each figure represents the mean of four replicate

Table 2:	Biofumigation efficacy of three previous crops on root rot disease of sesame plants during 2017 and 2018 seasons
*Means in column followed by the same alphabetical letter are not significantly different at 5% level according to LSD, *Each figure represents the mean of four replicates*

However, in 2018, biofumigation with both cauliflower and radish gave similar results after 80 days of transplanting. The disease severity index was 2.0 for both treatments. There were no significant differences between all tested treatments at (60 days after transplanting) in 2017.

The pesticidal effect of biofumigation has been attributed to the chemical breakdown products of glucosinolates (GLS), the characteristic constituents of brassica crops.

Isothiocyanates (ITCs) that have fungicidal properties are released in soil when GLS hydrolysis takes place among other secondary compounds^29-31 and Manici et al.³² showed that many Brassica species produce significant levels of glucosinolates (GLS), which are held in plant cells separately from the enzyme myrosinase and after chopping brassica plants and incorporating them into the soil in presence of water, the enzyme myrosinase is released and hydrolysis of GLS occurs producing the isothiocyanates.

Scientists ITCs toxicity, hence Brown and Morra²⁹ revealed that ITCs had the same effect of the active ingredient in the commercial fumigants dazomet and metham sodium and were highly toxic to pathogens. Similarly, Sarwar et al.¹² also observed that the Brassicaceae family suppresses pests and disease organisms. In general, this effect is attributed to a range of biocidal compounds that are released into the soil when glucosinolates are transformed into various bioactive fungicidal, insecticidal, nematocidal and herbicidal compounds.

In addition to the above mentioned pesticidal activity of glucosinolates, Many researcher have stated that incorporating large amounts of organic matter (Brassicas) into the soil also improved soil structure, increased nutrient availability, increased water holding capacity and stimulation of beneficial pathogen-suppressive microbial communities as reported by Kumar³³. In addition, Cohen et al.³⁴ confirmed that there is an inverse relationship between the presence of organic matter in the soil and plant root disease.

The differences in the influence of the species of Brassica on the pathogens attributed by Mithen³⁵. Kirkegaard and Matthiessen³⁶ and Sarwar¹² found that Brassicas are the most widely used plant species as biofumigants. The profile, concentration and distribution of different glucosinolates vary within and between Brassica species and in different plant tissues and consequently, the concentration and type of biocidal hydrolysis products evolved also varies³⁷.

CONCLUSION

The obtained data in this study demonstrated that brassica crops may be successfully applied as a safe and economical control measure for sesame charcoal root rot disease. Such feasibility of biofumigation crops may be improved by integrated disease management with other control measures for controlling charcoal rot of sesame.

SIGNIFICANCE STATEMENT

The current study demonstrated the positive and significant effects of applying biofumigation as a safe control measure that could be added to an integrated management program of charcoal rot disease of sesame crop. The results of this work could help other researchers to study biofumigation as a control measure that may help decrease our dependence on chemical fungicides and thus avoid the probability of developing resistance to those fungicides. The findings of this study may also provide insight into the mechanisms by which biofumigation effect soil-borne diseases. Also verified the potentiality of applying biofumigation as a biological control measure for soil-borne diseases at the field scale.