PATHOGENICITY OF FUSARIUM OXYSPORUM FROM DIFFERENT SOIL ENVIRONMENTS AND ITS EFFECT ON PHOTOSYNTHETIC ACTIVITY OF TOMATO PLANTS

Anna Wagner¹, Agnieszka Jamiołkowska², Władysław Michałek^3
1 Department of Plant Protection and Quarantine, University of Life Sciences in Lublin, Poland
² Department of Plant Protection and Quarantine, Faculty of Horticulture and Landscape Architecture, University of Life Sciences in Lublin, Poland
³ Department of Plant Physiology, Agricultural University of Lublin, Poland

ABSTRACT

Three populations of Fusarium oxysporum were tested for their pathogenicity to tomato seedlings. The isolates were collected from three soil environments of tomato: conventional cultivation (no cover crop), in field pea mulch and in rye mulch. Tomato plants of cv. Rumba were grown in a growth chamber in standard substrate with a pathogen inoculum prepared as described by Wagner (1997) or without the fungus. Pathogenicity was determined using the disease index, and the parameters of chlorophyll fluorescence were measured with PAM fluorometer. Results proved the effect of cover crops on F. oxysporum pathogenicity and some correlation between disease severity and photosynthetic capacity of tomato plants.

Key words: photosynthesis, cover crops, Fusarium oxysporum, tomato.

INTRODUCTION

Fusariosis induced by Fusarium oxysporum is one of the most severe diseases of tomato. The chemical control is not efficient or difficult to apply, therefore manipulating soil microbiota with cultural practices seems to be one of possible solutions [2]. Cover crops, recently applied by vegetable growers to suppress weeds and reduce production costs, can be an important factor in soil suppressiveness to pathogens [1, 9]. They affect fungal communities through root exudates and products of residue decomposition [6]. Previous experiments showed a significant effect of some cover crops on F. oxysporum populations in soil environment of tomato [7]. However, to determine soil suppressiveness it is necessary to know pathogenic abilities of population, as some strains of this fungus are non-pathogenic [3].

External disease symptoms not always show the degree of damage to plant physiology, therefore disease severity test of F. oxysporum collected from different soil environments was conducted together with chlorophyll fluorescence measurements. Chlorophyll fluorescence parameters help to evaluate the photosynthetic performance of plant, especially under environmental stress such as fungal infection [12]. There are many reports on plant diseases influencing photosynthetic activity of plant; however, our research is one of the first working with many isolates of different pathogenicity.

MATERIALS AND METHODS

Plants: Tomato seedlings, Lycopersicon esculentum Mill. cv. Rumba were grown in a growth chamber at 24-25° and 85% air humidity with 14-h photoperiod (from 6 AM to 8 PM). Sterilised seeds were first germinated in plates filled with sterile wood-wool covered with sterile blotting paper. When cotyledons appeared, they were transferred into pots containing sterile standard substratum (with pathogen inoculum or without). Plants were fertilised with Hoagland solution twice a week.

Inoculum: The analyzed isolates of F. oxysporum were obtained from the rhizosphere of tomato grown in three different environments: conventional cultivation (no cover crop), in field pea mulch (Pisum arvense L.) and in rye mulch (Secale cereale L.) without tillage. For each environment 13 isolates were tested. The inoculum of each isolate was prepared according to Mańka method described by Wagner [18]. The inoculation was conducted in 0.5 l pots filled with sterile substrate up to 3 cm below the edge. The slices of PDA overgrown with mycelium of F. oxysporum were put on the substrate, the seedlings were placed on the fungus and covered with substrate. For each combination 20 seedlings were tested. In the control combination PDA slices without pathogen were used. There were 4 replications in each combination.

Chlorophyll fluorescence measurements: After four weeks of plant growth in the pots, the parameters of chlorophyll fluorescence (F₀’, F_m’, ΔF/F_m) were measured with PAM 2000 fluorometer (Walz GmbH, Germany). For these tests, living leaves with the same orientation to light and the same position on plants were selected.

Estimation of disease severity: On the same day as chlorophyll fluorescence measurements, the seedlings were counted and evaluated for the disease severity, using 5-degree scale: 0 – no symptoms; 1 – small necrotic spots on lateral roots, no symptoms on leaves or stem; 2 – necrosis on all lateral roots; 3 – necrosis of tap root and stem base, wilting or chlorotic lower leaves; 4 – wilting of plant, completely rotten roots and stem base. Then the number of seedlings in each degree was computed. The disease index was estimated for each replication using McKinney’s formula [11]:

Disease index = Σa / Σb

where a = sum of products of numerical scale index (infection degree) and corresponding number of plants and b = total number of tested plants multiplied by the highest numerical scale index.

Afterwards mean disease index was computed for each combination. To check if the symptoms were caused by the pathogen, the fragments of affected stems and roots were placed on PDA after sterilising in 0.1% sodium hypochlorite and rinsing three times in sterile distilled water. After 7 days of incubation in 23°C the growing colonies were identified.

The results of pathogenicity tests and ΔF/F_m were analysed with Duncan’s test.

RESULTS

The symptoms on stems and roots were characteristic of F. oxysporum f. sp. radicis-lycopersici [17]. Wilting was related to stem and root rot, as the analyzed seedlings did not show typical tracheomycosis symptoms. In all combinations with pathogen isolates there were seedlings with necrosis on underground organs. However, the disease index for three isolates (R10, R7 and P1) did not differ significantly from the control (tab. 1). Only small necrotic spots were visible on lateral roots in these combinations. The most pathogenic proved the isolates P13, K6, R11, K7, R12, K12, R5, K4, K13, K9 and K10 (disease index > 60%). Most plants in these combinations were stunted, with yellowing, reduced leaves and all roots necrotic. Three isolates (K13, K9 and K10) caused wilting and damping-off. The disease index for other isolates ranged from 25% to 59%. Among them some (R9, P5, R2, R13 and K3) caused leaf size reduction and necrosis of tap root and stem base. Generally, the isolates obtained from the rhizosphere of tomato grown conventionally proved to be the most pathogenic. More than 50% of them were in the group of the highest disease index. Only one isolate from this field (K1) had the disease index < 40% (tab. 1).

The results of pathogenicity tests were confirmed to some degree by chlorophyll fluorescence measurements. The values of minimal (F₀) and maximal (F_m) yield were lower in all combinations with the pathogen. The effective quantum yield of PSII (ΔF/F_m) was also reduced. However, the differences were less significant than in the pathogenicity tests. The values of ΔF/F_m differed significantly from the control only for the seedlings with disease index > 46%, except for the seedlings inoculated with the isolates K1 and R6 (disease index 39% and 42%, respectively). The correlation between the disease index and fluorescence parameters was strong for seedlings inoculated with isolates of higher pathogenic abilities (K7, R12, K12, R5, K4, K13, K9, K10) (tab. 1).

DISCUSSION

The biodiversity is noticeable particularly among such facultative pathogens as F. oxysporum. The results of the tests indicated differences between the analyzed isolates what confirms other reports [3, 18]. Pathogenic abilities of fungi depend not only on their genomes but also on environmental factors. It was proved that root exudates influence the ability of Fusarium spp. to infect and damage plant tissues [16]. Also, some cover crops provide nutrients and improve soil structure what results in better plant condition [19]. Our test showed some correlation between the origin of isolates and their pathogenic abilities. The most pathogenic were the isolates obtained from the field without cover crop. Relatively the least pathogenic were the isolates from the combination with field pea mulch. This effect of the crop belonging to Leguminosae family can be explained by several hypotheses. Field pea could affect directly the pathogen through its metabolites exuded to soil when the plants were still alive and as mulch through the products of its decomposition [6]. Root exudates and decomposition products stimulate the development of soil microbiota that could affect with their metabolites the pathogenicity of F. oxysporum. Some saprotrophic fungi and bacteria are able to act as plant growth promoting organisms inducing plant systemic resistance [4, 8]. Also nutrients derived from field pea could favourably increase the resistance of tomato plants in the field what can result in decreasing F. oxysporum pathogenicity. The population of pathogen isolated from the rhizosphere of tomato grown in rye mulch was more diversified in its pathogenic abilities. Rye is considered a good phytosanitary crop but probably its effect on pathogens is stronger as living crop than as mulch [6].

Many authors [5, 10, 13] report the decrease of fluorescence parameters under stress caused by pathogen infection. When pathogens of green organs can affect the photosynthesis directly, root infection influences the process by interfering with several physiological processes what results in the inhibition of photosynthesis [10, 15]. The changes were more evident in values of ΔF/F_m, showing decreasing efficiency of PSII photochemistry. However, the correlation between disease index and chlorophyll fluorescence parameters was particularly evident for plants infected in the highest degree but not so significant for other plants. It could be partly explained by the age of plants. The seedlings were only four weeks old and according to Nogues et al. [13] the changes in phytosynthesis process are more pronounced in older plants.

Two isolates (K1 and R6) caused more evident decrease of ΔF/F_m than other isolates with similar or higher pathogenicity. It can be supposed that the pathogen colonised not only root cortex but also the vascular system, affecting water transport in the plants and resulting in the decrease of effective quantum yield of PSII [13]. Even if F. oxysporum f.sp. radicis-lycopersici is regarded mostly as cortex pathogen, some strains can invade xylem, although the process is longer than in typical tracheomycosis [17]. It is difficult to prove because no hyphae were found in vascular tissues, however, the changes in xylem can be caused by fungus metabolites. The isolate P8 did not affect ΔF/F_m, even if the disease index amounted to 48%. In this case, probably the symptoms were only superficial and the pathogen infected only the external cortex.

The results of investigations of cortex specific pathogens and their effect on photosynthesis are more difficult to interpret than those of wilt pathogens are because several mechanisms can be involved both in pathogenesis and in changes in photosynthetic activity of plant. Apart from direct influencing leaf size or water movement, the disease process also induces defence mechanisms in plants what can also affect the process of photosynthesis [14]. The presented results contain only some parameters. To be able to learn more about the correlation between pathogenic abilities of root pathogens and photosynthetic activity of infected plants, other chlorophyll fluorescence parameters as well as gas exchange and chlorophyll content should be determined.

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Accepted for print: 16.01.2007

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