Corms were surface-disinfested with 20% sodium hypochlorite for 30 min, followed by three rinses in sterile distilled water a wound 1-2 mm deep aseptically made with a cork borer (10 mm in diameter). A mycelial disk of the same diameter, taken from 7-day-old potato-dextrose-agar culture, was placed in the wound. A disk of sterile PDA was used for the control corms. Inoculated corms were placed in containers containing sterile filter paper No1 and maintained in incubator at 24 degree C, 80% RH. The experiment was repeated twice.
Physiological studiesTemperature To determine the effect of temperature on fungal growth, 10-mm-diamter disks were transferred from F.oxysporum f. sp. gladioli culture edges and placed on 90-mm-dimeter Petri dishes containing PDA. The linear hyphal extension rates (cm2/day) of F.oxysporum f.sp. gladioli fungal isolates were determined between 5 and 35ï¿½C on PDA (10 ml) in 9 cm-diameter Petri plates. For each treatment, Five replicate plates were inoculated with PDA discs (10 mm-diameter) taken from the edge of actively growing cultures with a sterile cork-borer. The plates were sealed with Prafilm(Japan, Co.).
PH. The effects of pH on biomass production and spores production of the three fungal isolates in Zapacks[A3] medium were determined in liquid culture using a Zapadoks[A4] medium already known to support mycelial growth of the three fungal isolates. Appropriate amounts of citrate phosphate buffer .The pH ranges were adjusted between 4 and 8. The original pH of unbuffered medium was 4.5. Medium was sterilized by autoclaving at 120C and 103.4 kPa for 20 min.
Five replicate of Erlenmeyer flasks (250 ml) containing 100ml of Zapacks[A5] medium were inoculated with 1 ml fungal spore suspension containing 1 x lo6 conidia taken from fungal colonies grown on PDA. After 7 days. The cultures were incubated at 25o C on rotary shaker 250 rpm for 7 days. The mycelial mats were harvested by vacuum filtration on to pre-weighed filter papers (Whatman No. 4) and oven-dried for 48 h at 80ï¿½C. spore concentrations were counted using a haemocytometer under a light microscope at a magnification of 40.
Water potential (aw). The water potential of PDA was adjusted osmotically with the non-electrolyte glycerol. The matric potential of PDA was adjusted with polyethylene glycol 6000 (PEG). In addition, PDA [39 g Potato dextrose agar (Oxoid L13) in1 litre distilled water] was osmotically adjusted by the addition of glycerol only. Quantities of either glycerol, were added to the media to give required water activity in the range of 0.96, 0.95,0.94,and 0.aW. The matric potential was adjusted using the following formula aw = n1, moles of solvent and n2, moles of solutes
The Medium was sterilized by autoclaving at 120ï¿½C and 103.4 kPa for 20 min. The PDA media was poured in to the plates 20 ml/ plate. After solidification the plates were inoculated with PDA discs (10 mm-diameters) taken from the edge of actively growing cultures with a sterile cork-borer. The plates were sealed with Prafilm(Japan, Co.). Five replicates were prepared for each.
Growth media. To study the effect of different nutrients on F.oxysporum f. sp. gladioli strain G010 growth. Different media were employed in this study to know which media will maximize the growth of F.oxysporum f. sp. gladioli G010 strain. Five ready prepared media were studied Potato dextrose agar, Nutrient agar, Malt extract, Zapax, and Corn meal (Sigma, Co. UK). The media were prepared according to manufacture recommendations. Data were recorded after 7 days from inoculation under 220 C.
Statistical analysis All experiments were conducted twice. Treatments were organized as a complete block design, Statistical analyses were conducted using the general linear model procedures of the statistical analysis system (SPSS version15). Experiments were analyzed using standard analysis of variance (ANOVA) with interactions. Significance was evaluated at P<0.05 for all tests. Mean separation was accomplished using Tukey HSD test. Since repeated tests yielded similar results, data from single representative experiments are presented.
Results Growth rate differences Differences in growth rate among F.oxysporum f. sp. gladioli isolates were substantial (P< 0.001; Fig. 1) .F.oxysporum f. sp. gladioli G010 has faster growth rate than other isolates. No difference in the growth of 640 and 160 strains. Data showed that 640 and 160 strains were lower than G010 strain.
Physiological studies Temperature Differences in growth rate of F.oxysporum f. sp. gladioli strains were found at the different incubation temperature P<0.001.The temperature assay revealed that there was difference in strains response to the different incubation temperature (Fig. 2, A, B, C). G010, the optimum growth rate was found between 20-250 C. How ever 640 and 160 strains, the optimum growth was found within 25-300 C. No growth found at 40 C and 350 C. 640 and 160 strains growth was higher at 150C compared with G010.
pH Difference in growth rate among F.oxysporum f. sp. gladioli were detected at different pH levels P<0.001 (Fig. 3). The optimum pH for G010 was 8, and 5, 4 for 640 and160 respectively. But the growth rate was slower at pH 6 and 7.No growth was observed for G010 at pH 4. The same trend was obtained with the number of spores (Fig. 4). Water activity (aw): F.oxysporum f. sp. gladioli (G010) strain continued to grow normally at different aw. However, the Different potential water activity reduced F.oxysporum f.sp. gladili growth rate compared with the control with no difference. The growth rate was beaked after 2 days and began to reduce linearly( Fig. 4).
Growth media Difference in F.oxysporum f. sp. gladioli was monitored on different growing media( P<0.001, Fig. 5). The optimum growth was obtained with Potato dextrose agar media, followed by Nutrient agar media. While, Corn meal agar was the lowest media suitable for F.oxysporum f. sp. gladioli. Discussion Despite being genotypically uniform F. oxysporum f. sp. gladioli strains proved to be phenotypically diverse. The consequence from this finding is important and it means the variation in their virulence, culture morphology and physiological requirements. The ability of some isolates to grow at different temperature and pH regimes make F.oxysporum strains flexible pathogen, and explain their ability to survive under different environmental and production conditions( Bhatti, 1992).
Difference in F.oxysporum f. sp. gladioli strains growth rat could happen for many reasons; it could happen because there is a difference in isolation source. The studied strains were isolated from different sources. G010 was isolated from the purchased corms, 640 strain was isolated from infected soil in Netherlands, and 160 strain was isolated from infected soil as well but from Germany. This mean they are genotypically diverse and their growth rate normally will be different and their Pathogenity will differ as will.
It hase been well documented that. The temperature is important in the progress of F. oxysporum invasion and symptoms development (Beckman et al., 1962). The maximum distribution of the pathogen in the viscular system and subsequent development occurred at 26O C soil temperature ( Ploetz et al., 1990).How ever Foc symptoms became most obvious in spring and early summer( Viljoen 2002).
The optimum cultural growth temperature for Foc in this study was 25o C. Similar results were reported for F.oxysporum f. sp. fabae ( Ivanovic et al., 1987) and F.oxysporum f. sp. spinaciae ( Naiki and Morita 1983). But the growth was limited below 15oC,Brake et al., 1995. Another reason for B. brevis inefficiency could be affected by Gladiolus corms root exudates, Weller 1987 reported that bacterial root colonisation is affected by host genotype[A6].