Effect of temperature and pH on in vitro growth rate and sclerotial density of Colletotrichum coccodes isolates from different VCGs

American Journal of Potato Research, Sep/Oct 2003 by Nitzan, N, (Lahkim), L Tsror

ABSTRACT

The radial growth rate and sclerotial density of Colletotrichum coccodes isolates representing four major vegetative compatibility groups (VCGs) were determined at three temperatures (21, 25, and 30 C) and three pH levels (pH 5, 6, and 7). This is the first report indicating that C. coccodes isolates characterized to the same VCG share common physiological traits. The optimal temperature for growth of isolates from all four VCGs was 25 C, except for VCG1 isolates, for which growth at 21 and 25 C was equivalent. The growth rate of all isolates, of all VCG groups was decreased at 30 C, whereas sclerotia development was enhanced. The optimal pH level for growth of all isolates was pH 6 and pH 7. Interactions between VCGs and temperature or pH were observed in relation to radial growth rate and sclerotia density. At 25 C, isolates from VCG1 and 2 demonstrated the most rapid growth rate. At 21 and 30 C, VCG1 isolates demonstrated the fastest growth compared with all other VCGs. In response to pH, VCG1 and 2 demonstrated the fastest growth rates at all pH levels. VCG3 and 4 demonstrated the highest sclerotial density at 25 C and at pH 6; and VCG2 and 4 demonstrated the highest sclerotial density at 30 C.

Accepted for publication 27 June 2003.

ADDITIONAL KEY WORDS: Vegetative compatibility, Black dot, potato.

INTRODUCTION

Colletotrichum coccodes (Wallr.) S.J. Hughes, the causal agent of black dot on potato, may cause up to 30% yield reduction in susceptible cultivars, and damage tuber quality (Andrivon et al. 1997; Denner et al. 1997; Tsror et al. 1999). The fungus is classified to the sub-division Deuteromycotina, lacking a known sexual cycle, where vegetative compatibility may serve as a means of genetic exchange among its isolates. Vegetatively compatible isolates are able to anastomose and form a stable heterokaryon, creating genetically isolated sub-populations referred to as vegetative compatibility groups (VCGs) (Leslie 1993). Isolates assigned to the same VCG tend to share more common traits compared with isolates from different VCGs. Therefore, members of different VCGs may differ morphologically, physiologically and pathologically (Leslie 1993).

In a previous study, we characterized four major VCGs among 110 Israeli, French, and Dutch isolates of C. coccodes collected from potato (Nitzan et al. 2002). Thirty-four representative isolates from the four major VCGs were tested for their aggressiveness to potatoes in environment-controlled growth chambers. Isolates from VCG3 were the most aggressive to potato, as expressed by high amounts of stem colonization levels and high densities of sclerotia on roots and crowns, suggesting that there is significant VCG diversity in this species and that it may be correlated with pathogenic characteristics / specialization (Nitzan et al. 2002).

Possible correlations between VCGs and physiological traits were reported previously. VCG2 isolates of C. dematium f. sp. spinaciae, grown on ALBA (acidified lima bean agar) medium demonstrated a yellow pigment, whereas VCG1 isolates did not demonstrate any particular pigmentation (Correll et al. 1993). Similar results wore observed with Verticillium tricorpus, where isolates defined as VTR1 and VTR2 demonstrated a medium yellow pigment, while isolates in VTR3 demonstrated a dark yellow pigment, and those characterized as VTR4 demonstrated a light yellow pigmentation (Korolev and Katan 1998).

In the present study we hypothesize that C. coccodes isolates assigned to different VCGs possess different physiological traits. The specific objectives were to examine the effects of temperature and pH on in-vitro growth rate and sclerotial density of C. coccodes isolates from the four major VCGs.

MATERIALS AND METHODS

Fungal Isolates

Thirty-eight randomly selected representative isolates of C. coccodes from the four major VCGs (Nitzan et al. 2002) were used in the present study. Of the 38 isolates, 26 were unique to the temperature experiments, two were unique to the pH experiments, and 10 were common to both sets of experiments. In the pH experiments we used three isolates from each VCG (total of 12 isolates), and in the temperature experiments we used four, 14, nine and nine isolates from VCG1, 2, 3, and 4, respectively, which comprise 50%, 36%, 36%, and 82% of each VCG. The isolates were originally obtained from infected potato seed tubers from The Netherlands and France, and from infected potato plants and tubers from Israel. The isolates were sub-cultured on potato dextrose agar from cool-stored cultures before use.

Colony Growth Rate

The radial growth rate of the representative C. coccodes isolates was determined in three temperatures: 21 C, 25 C, and 30 C, and three levels of pH: 5, 6, and 7 (pH was adjusted by either 1M NaOH for pH 6 and 7, or 1M HCl for pH 5). In the temperature experiments the pH of the medium was 5.6, and in the pH experiments the temperature was 25 C. One-millimeter plugs of mycelium from 7-day-old colonies were plated onto 1% PDA (Difco Laboratories, Detroit, MI, USA), in the center of a petri dish (9 mm), and incubated in the dark. Experiments were repeated three times with five replicates per each isolate. Multiple measurements at right angles of colony diameter were done every 48 h for 10 days (five measurements), and the growth rate was calculated using linear regression.