Study of the Differentiation of Fusarium oxysporum f.sp. albedinis Chlamydospores on Different Culture Media

Chlamydospores of Fusarium oxysporum f.sp. albedinis are well known as fungus survival spores in soil, also act as a causal agent of Bayoud disease of the date palm. Till now, there are a little study in the literature about the favorable media for the formation of chlamydospores and the stages of chlamydogenesis. This study shows that rice flour based medium (45 chlamydospores per mm 2 ), oatmeal (31 chlamydospores per mm 2 ) and bean flour (27 chlamydospores per mm 2 ) tested for the first time, are among the media inducing the formation of chlamydospores in large numbers. The optimum formation of chlamydospores is observed on rice flour-based medium after 7 days of Original Research Article incubation at 18°C and pH 7. The effect of light does not seem very important on chlamydogenesis. However, relative humidity has a significant effect on the growth and chlamydogenesis of F. oxysporum f.sp. albedinis. The tested G 1 isolate grows and forms chlamydospores at relative humidities of 70 and 100%. The number of chlamydospores varies between 40.01 and 44.05 chlamydospores/mm 2 . To form chlamydospores, cultures of F. oxysporum f.sp. albedinis need to be in contact with the favorable culture medium. Thus, the cultures develop and form more chlamydospores on a single layer of cellophane deposited on a culture medium, but show slower growth and do not form chlamydospores on two layers of cellophane. Chlamydospores of Fusarium oxysporum f.sp. albedinis are always at the extremity of the mycelial filaments. They differ in one to four cells in superimposed position. A terminal hyphal bulge leads to the formation of the terminal cell. The second cell is formed at the base of the first, and the process continues so that one can sometimes observed chlamydospores in bead-like chains.


INTRODUCTION
For more than 100 years, the palm groves of Morocco and Algeria have been devastated by a tellurium fungus, Fusarium oxysporum f.sp. albedinis, which causes rapid decline of the date palm [1]. This vascular fusariosis, commonly called Bayoud, has decimated most commercial varieties [2].
F. oxysporum f.sp. albedinis produces three types of asexual spores [3,4]. These are microconidia with variable size (3-15 x 3-5μm) and are often uni-cellular, sometimes bi-cellular, and rarely have two partitions [5]. Macro-conidia measuring 20-35 x 3-5 μm with pointed and short extremity. On cultures, in soil or on infected palm fragments, even in the absence of its host, the fungus produces elements of resistance which are chlamydospores either from the macroconidia or from the mycelial cells which can be found isolated or in pairs, rarely in chains [6].
Many authors have given a detailed description of the macroscopic and microscopic characteristics of life cycle components of F. oxysporum f.sp. albedinis [7,8,9]. But, there was study neither on favorable culture media for the formation of chlamydospores nor the process of chlamydogenesis.
In this study, different culture media were tested to determine the most favorable medium for the formation of chlamydospores of F. oxysporum f.sp. albedinis, forms of fungus survival in soil and on plant debris. In addition, the steps of formation of these chlamydospores were also studied.

Isolation of Fusarium oxysporum f.sp. albedinis
The fungus can be easily isolated from date palm roots exhibiting typical symptoms of Fusarium wilt [10]. Seven isolates of F. oxysporum f.sp. albedinis were isolated from the roots of the date palm from the Ziz valley in Errachidia (Isolates: 64, 42, 51, 124, 93, and 97) and a single G1 isolate from the Goulmima region in southeastern Morocco.
The roots were disinfected with alcohol and then dried on sterile filter paper before being deposited on PSA medium (Potato-Saccharose-Agar: 200 g of potato; 20 g saccharose; 15 g agar; 1000 mL distilled water). The cultures obtained after incubation for seven days at 28°C were purified by successive subcultures and preserved for subsequent studies.
For each culture medium, three Petri dishes were seeded with an explant of 5 mm diameter, taken at the margin of a young culture of seven days. After seven days of incubation in the dark and at 28°C., sporulation was evaluated by taking five 5 mm diameter discs. These discs were put into a test tube containing 1 mL of tap water and agitated with a vortex to get free chlamydospores.
About 0.1 ml of the suspension was mounted on a glass slide and the number of spores was counted with respect to the microscopic field. Only culture media that promoted the production of a large number of chlamydospores was maintained for further testing.

Influence of Rice Flour-based Medium on the Induction of Chlamydospores in Different Isolates of Fusarium oxysporum f.sp. albedinis
Six isolates of F. oxysporum f.sp. albedinis collected from the roots of the date palm from the Ziz Valley and a single isolate from the Goulmima region were tested on rice flour based medium (14 g rice flour; 4 g yeast extract; 15 g agar; 1000 mL distilled water).
Three Petri dishes were seeded with an explant of 5 mm diameter taken at the margin of a seven days young old culture on the PSA medium. The dishes were incubated 24-hours in the dark at 28 C. Three repetitions were performed for each case. Only isolates that produced a large number of chlamydospores would be maintained for further testing.

Influence of the Succession of Two Culture Media (PSA and Rice Flour) in the Same Petri Dish
In a sterile Petri dish, two culture media were prepared in two concentric rings separated by two sterile cylindrical cardboards lined with waterproof membrane to isolate the culture media. The first smaller ring was filled with rice flour by carefully incorporating under sterile conditions a cylinder which delimited the inner core. Once the medium had solidified, a second wider cylinder was introduced and the space between the first and the second cylinders was filled with PSA medium. At the end, the space between the second cylinder and the Petri dish was filled again with the medium made from rice flour ( Fig. 1).
A cut disc of the mycelium, taken from a young culture of the fungus (isolate G1), developed on the usual PSA medium, was transplanted in the center of this Petri dish containing different culture media in concentric rings.

Effect of pH and temperature on chlamydogenesis
A pH range (4, 5, 7, 9, and 12) was obtained by adding HCl or NaOH [1N] to the rice flour medium. The pH was adjusted before autoclaving. Similarly, a temperature range (5°C., 18°C., 37°C., 44°C.) was studied on a medium based on Rice flour. Three replicates were performed. The Petri dishes were seeded and incubated in the same manner as previously.

Effect of light on chlamydogenesis
Petri dishes containing the rice flour medium were seeded by mycelial discs of a young culture of F. oxysporum f.sp. albedinis developed on PSA. Three dishes were incubated in the dark and three others exposed to light. Three replicates were performed.

Effect of relative humidity on chlamydogenesis
To assess the effect of relative humidity (RH) on chlamydogenesis, we used the technique of Wash-burn (1982). This technique consists of the use a mixture of distilled water and glycerine at different RH values: For RH = 0 (100% glycerine); For RH = 30 (30% distilled water and 70% glycerin); For RH = 50 (50% distilled water and 50% glycerin); For RH = 70 (70% distilled water and 30% glycerin); For RH = 100 (100% distilled water); 10 mL of these solutions were distributed in Petri dishes.

Fig. 1. Diagram of a petri dish containing two concentric culture media
Sterile slides, bearing a layer of rice flour medium (one layer per slide), were inoculated by cuttings of a young culture of F. oxysporum f.sp. albedinis taken from a PSA plate. These slides were then placed on sterile beads in sterile Petri dishes. Three replicates were made for each RH.

Formation of chlamydospores on cellophane membrane
The nature of the substances inducing the formation of chlamydospores was demonstrated by the technique of cellophane [11,12]. Three Petri dishes containing the rice flour culture media were covered with a layer of sterile cellophane, and three other dishes containing the same culture medium were covered with two sterile layers of cellophanes. Discs of 5 mm in diameter from a 7-day old culture of Fusarium, were placed in the center of the surface of the cellophane. The dishes were incubated for 24hours. At the end of this time, the adhering mycelium was removed and analyzed under an optical microscope.

Effect of heat shock on germination of chlamydospores
Each of the Test tubes contains 1 mL of the suspension of chlamydospores from F. oxysporum f.sp. albedinis were exposed for one hour at temperatures of 5°C, 40°C, 50°C, 56°C, and 60°C. For each temperature, one drop of the suspension was spread on Petri dishes containing the PSA medium. The dishes were incubated seven days in the dark and at 28°C.

The Stages of Chlamydogenesis
Three 5 mm diameter discs from a seven-day old culture on Rice flour medium were placed in a test tube containing 1 ml of tap water and one drop of cotton blue, The mixture was milled and then vortexed. A 0.1 mL drop of the suspension was placed between the slide and the cover slip.

RESULTS AND DISCUSSION
On rice flour, only isolates G1 and Isolate 64 from F. oxysporum f.sp. albedinis formed chlamydospores, with 46 and 2 chlamydospores/mm 2 respectively. The G1 isolate was also able to form chlamydospores on oatmeal, bean and celery media.
The rice flour medium, most favorable for the formation of chlamydospores, was used [13] to induce the rapid formation of chlamydospores in certain pathogenic yeasts, e.g. Candida albicans.
The celery medium did not allow the formation of chlamydospores, contradictory to that reported previously by Huang [14]. This author studied the effect of plant extracts on the induction of chlamydospores in Fusarium oxysporum and other species of the Fusarium genus and noted that the celery-based medium was the most favorable for chlamydospore formation. In Fusarium solani, the formation of chlamydospores was observed after incubation of the cultures on PDA at 25° C. for 10 days or, according to the method of Smith and Snyder [15], after an external supply of carbon and nitrogen [16] or after addition of glucose to a solution of the soil [17]. Carbon sources, mainly citrate, and a pH ranging from 5.2 to 6 also promote the production of chlamydospores [18]. Other authors have reported that some herbicides stimulate the formation of chlamydospores in F. oxysporum, as in the case of glyophosate, at a concentration of 1.5 mM [19]. Ciotola et al. [20] noted that the extract of fertilized straw of Striga hermonthica induces the formation of chlamydospores by mass in F. oxysporum after incubation of the cultures for 21 days at 21°C. or 14 days at 30°C. Similarly, strains of Phytophthora palmivora (K and 1) were able to form a large number of chlamydospores on oatmeal medium and much less on pea media, coconut milk, and synthetic media supplemented with amino acids [21]. Chlamydospores were observed on the media well before the depletion of the reserves, implying that elements of these media induce their formation. In Cryptococcus neoformans, responsible for meningoencephalitis in humans, the formation of chlamydospores was closely associated with the production of aflatoxins [22]. Alicia et al. [31] reported that cultures of some strains of C. albicans, incubated at 28°C, sporulated and formed chlamydospores in a shorter time (16 hours) on CMB synthetic medium (cornmeal broth) supplemented with 5% of milk, while Bennet et al. [32] reported that the production of chlamydospores in F. oxysporum f.sp. vasinfectum is rapid on supersol broth medium without glucose.
The succession of culture media within the same Petri dish influences the formation of chlamydospores in F. oxysporum f.sp. albedinis (Fig. 3). Indeed, after incubation for seven days in the dark, the fungus growing on the centre of rice flour medium plate formed chlamydospores. On the other hand, the fungus on PSA medium, did not form these structures but when the mycelia reached the edge of the dishes containing the rice flour medium chlamydospores were produced. The pH and temperatures also influenced chlamydogenesis in F. oxysporum f .sp.albedinis. In acid medium, cultures growth was almost null and chlamydospores did not form. In a neutral medium (pH in the order of 7), the mycelial growth was maximum and the optimum rate of the chlamydogenesis was observed (  [33] reported that the formation of chlamydospores in C. albicans is highly dependent on the pH of the culture medium. When the medium is basic, mycelial growth (MG) is much greater (6 and 7 cm) than that observed in a neutral medium (4 cm), but the chlamydogenesis is low at pH 9 and becomes null at pH 12. For a given column, the values followed by the same letter do not differ significantly at the 5%.

Culture medium
The maximum rate of the chlamydospores formation is observed on rice flour-based medium after 7 days of incubation at 18°C. in the studied isolate (Table 2). Above 37°C, fungus showed very slight growth and chlamydospores were absent. Bulit et al. [34] reported that the optimum temperature for growth of F. oxysporum f. sp. albedinis (Foa) is between 27° and 28°C and mycelial growth is inhibited at around 38°C. For a given column, the values followed by the same letter do not differ significantly at the 5%.   For a given column, the values followed by the same letter do not differ significantly at the 5%. According to Dennis and Webster [12], and Berber et al. [11], certain substances of the culture medium were necessary for the induction of chlamydospores. The use of a single layer of cellophane allows the diffusion of these substances, but two layers of cellophane make it a barrier to this diffusion. The cellophane technique made it possible to isolate the chlamydospores and to test their germinative ability after a thermal shock. These resistant structures retained their germinating power after a one-hour heat shock in the range of [5°C., 50°C.]. Beyond 50°C, chlamydospores lost their germinative power (Table 2). Bounaga [35] reported that the chlamydospores of F. oxysporum f.sp. albedinis lose their germination capacity at a temperature of 60°C.

CONCLUSION
The in vitro formation of chlamydospores of F. oxysporum f.sp. albedinis varies depending on the tested media culture, the incubation temperature and the pH of the medium. The rice flour-based culture medium is the most favorable for chlamydogenesis; it far exceeds the effect of the media based on flour of Avoine, beans and the one based on plaster. The optimum production of chlamydospores is observed in the G1 culture growing on rice flour medium, after 7 days of incubation, at 18°C. and pH 7. Under these conditions, the number of chlamydospores is important for relative humidities of 70 and 100%. The current study provided new information concerning their differentiation on the stages of chlamydogenesis in F. oxysporum f.sp. albedinis.