Harling et al., 1988 suggested that temperature alter the balance between the host plant and Fusarium wilt pathogen.The effect of temperature on wilt occurrence may vary in different pathosystems( Ben-Yephet and Shtienberg 1997). Brake et al., 1995 found that the impact of temperature was on plant growth not on the pathogen. F.oxysporum f. sp. cubense is present in some tropical regions, it severly affects banana in the subtrobics, indicating that temperature may have an important influence on disease development ( Ploetz et al., 1990).
Ph
Increasing soil pH has appositive effect on Fusarium wilt disease reduction (Jons and Woltz 1973). The explanation for this effect are, The domination of advantageous and disease –suppressive in the root rhizosphere at high pH. And the effect of pH on micronutient availability in the media and its impact on the growth , sporulation and virulence Jones et al., 1989. F.oxysporum mycelia grow within range of pH 2-12, how ever F.avenaceum started to grow at pH 3. But all species were observed to grow at pH 6, Srober 1978).Wilson,1946,found that acid soil (pH4.2) supported Fusarium growth, whereas a pH near neutrality prevented this growth .Raising soil pH toward neutrality appears to be a foundation in controlling Fusarium wilt, which is a commonly disease associated with acidic conditions ( Woltz and Jones 1981). F.oxysporum spores germination were influenced by the difference in the pH (Peng et al. 1999)
Nutrition
The stages of Fusarium growth depend on the ecological balance and nutrient availability ( Woltz and Jones 1981).Fusarium oxysporum is an autotroph, requiring only a carbon source for structure and energy, and inorganic compounds to synthesized organic compounds such as sugars, lipids and amino acids ( Woltz and Jones 1981). Increasing nitrogen levels in the soil generally lead to an increase in Fusarium wilt development (Woltz and Jones 1973) Ammonium form of nitrogen becomes more favourable for Fusarium disease with increasing the rate of application( Woltz and Jones 1973), and reported also F.oxysporum cultured on ammonium nitrogen was more virulent than the cultured on nitrate nitrogen form.This work was aiming to understand the physiology patterns of F.oxysporum f. sp. gladioli strainsthat regulate the fungus virulence.
The aim of this was is to investigate and gain more understanding for physiological factors affecting F.oxysporum f. sp. gladioli growth.
Materials and methods
Fungal isolates
F.oxysporum f.sp. gladioli strains ( 640, 160) were purchased from , Netherland, but Go10 strain s was obtained from a Gladiolus Big Flower GT01 corm. Cultures were maintained on PDA (Oxoid, Basingtoke, Hants, UK) at 22◦C and routinely sub–cultured at 15 day intervals, to be used as a fresh stock cultures for sub-culturing. 640 and 160 strains were delivered as freeze dried spores. Spores were suspended in 1 ml sterilitiezed water and cultured on PDA plates for 15 days. Cultures were maintained on PDA (Oxoid, Basingtoke, Hants, UK) at 22◦C and routinely sub–cultured at 15 day intervals, to be used as a fresh stock cultures for sub-culturing. Subcultures were prepared by inoc6lating PDA with 1 cm diam. disks of colonized PDA plus mycelium, cut from the edge of an actively growing, 7 day old colony. The new plates were incubated at 25O C for 7 days. The 10-mm disk diameter was subtracted from this value. Five plates were incubated for each treatment and the experiment was repeated.
Fusarium oxysporum f.sp. gladioli strains growth area were determined by monitoring the daily growth between the isolates.
Pathogenity test
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 studies
Temperature
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 35°C 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 medium were determined in liquid culture using a Zapadoks 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 120°C and 103.4 kPa for 20 min. Five replicate of Erlenmeyer flasks (250 ml) containing 100ml of Zapacks 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 80°C. 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 120°C 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.
Fusarium disease considered to be associated with acidic sandy soils rather than heavier soils with higher pH walues( Woltz and Jones 1981). The results showed that the different strains responded diversely fro different pH levels. This could be due to
Howie et al., 1983 studied the effect of rhizosphere matric potential on Wheat –root colonised by Pseudomonas fluorescens, the results indicated 0.3-0.7 bars was the range in which oxygen availability and turgor potential of the cells and nutrient availability were optimal for bacterial –cell growth.
This work consider one of the steps which had taken to understand he differences Between F.oxysporum f. sp. gladioli strains. But this work happened without knowing enough information about the history of the strains for example the growing crops in the infected soil which those strains were isolated from.
The approach outlined in this study should be confirmed by molecular work to deeply understand the real difference between those straind
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Table 1
Table 2
Writ about water potential
complete and write about nutrition