PHYTOPATHOLOGY/ MYCOLOGY N. Boughalleb and M. E1 Mahjoub (2006) Phytoparasitica 34(2): 149-158

Fusarium solani f.sp. cucurbitae and F. oxysporum f.sp. niveum Inoculum Densities in Tunisian Soils and Their

Effect on Watermelon Seedlings

N. Boughalleb 1'* and M. E1 Mahjoub 1

Populations of Fusarium solani f.sp. cucurbitae ( Fsc ) and Fusarium oxysporum f.sp. niveum (Fort) in naturally infested soil of watermelon fields were counted by the soil dilution method with subsequent pathogenicity tests. Inoculum density varied within the same region from one field to another, ranging between 9 and 1600 CFU g-1 soil for Fsc and from 0 to 200 CFU g-1 soil for Fon. Fusarium crown- and root-rot-diseased seedlings were observed in most soils (93%); however, Fusarium wilt was observed in only 34% of soil samples. The disease incidence on cv. 'Giza' (Y) increased significantly with inoculum density in the soil (X) (P<0.001). For Fsc, the relationship between inoculum density and disease incidence was characterized by the equation Y=0.0005X+0.165 (R2=0.67). For Fon, the equation was Y=0.003X-0.0014 (R2=0.88). Based on these equations, the estimated inoculum densities required to cause 50% disease incidence (Dis0) on cv. Giza plants was 670 and 171 CFU g-1 soil for Fsc and Fon, respectively. KEY WORDS: Disease potential; Fusarium oxysporum f.sp. niveum; Fusarium solani f.sp. cucurbitae; watermelon fields.

INTRODUCTION

Crown and root rot diseases of cucurbits caused by Fusarium solani (Mart.) Sacc. f.sp. cucurbitae W.C. Snyder & H.N. Hans. (Fsc) severely damage watermelon. This pathogen was first reported on squash (Cucurbita pepo L.) in 1930 in South Africa (8) and subsequently on different cucurbits in the United States (13,25), Spain (1,11), Italy (10), New Zealand (14) and Japan (22). In Tunisia, Fsc has been found wherever watermelon has been grown and is a serious problem for its production. The E solani isolates collected from different Tunisian cropping areas were identified as Fsc races 1 and 2 on the basis of pathogenicity tests on watermelon seedlings and muskmelon fruits. Race 1 is widely distributed in watermelon production areas; race 2 has a lower incidence but is present in northern, central and southern Tunisia (3). Fusarium wilt of watermelon caused by Fusarium oxysporum Schlechtend.:Fr. f.sp. niveum (E.ESm.) W.C. Snyder & H.N. Hans. (Fon) is also an economically important disease of this crop, well established throughout the watermelon-growing regions of the world. It has been found in Italy (7), Israel (24), Florida (9,20), Oklahoma (6), Cyprus (16), Greece and Turkey (21), Spain (12), Texas (5,19), and Delaware and Maryland (28). It has also been detected in Tunisia, where three races of Fon (0, 1 and 2) were identified in watermelon-growing areas, with a prevalence of races 1 and

Received June 9, 2005; accepted Oct. 2, 2005; http://www.phytoparasitica.org posting Feb. 14, 2006. tEcole Sup&ieure d'Horticulture et d'Elevage, Chott Mariem 4042, Sousse, Tunisia. *Corresponding author [e-mail: bougtn@yahoo.frl.

Phytoparasitica 34:2, 2006 149

2 (4). These soilborne pathogens cause two of the most severe diseases of watermelon, and in many areas of Tunisia they have become a factor that limits its production.

There is no information about F. solani and Fsc inoculum densities in the soil of naturally infested watermelon fields. Some studies have determined the population of E oxysporum in soils cropped to watermelon. Netzer (24) estimated that the population of this fungus within heavily infested watermelon fields ranged from 400 to 1400 CFU g-1 soil. Within this range of inoculum densities, more than 95% of the seedlings of watermelon cv. 'Sugar Baby' wilted in a greenhouse bioassay (24). In Florida, Hopkins and Elmstrom (15) reported that populations of F. oxysporum averaged around 1500 CFU g-- 1 soil in most research plots cropped to watermelon. Nevertheless, little is known about the range and distribution of inoculum densities of Fon in infested commercial fields used for watermelon production. Recently, Zhou and Evens (29) detected inoculum densities of Fon ranging from 100 to 1200 CFU g-1 soil in Maryland. Disease incidence of susceptible seedlings of watermelon cv. Sugar Baby reached over 20% when seedlings were planted in samples of soil collected from fields exhibiting sudden wilt symptoms. In another study, Zhou and Everts (30) quantified root and stem watermelon colonization by Fon and the relationship to Fusarium wilt incidence, indicating that this pathogen is able to penetrate to all parts of susceptible watermelon cultivars, but the level of colonization varied with cultivar resistance.

The objectives of this study were to: (i) conduct a survey of inoculum densities of E solani and E oxysporum in watermelon fields in Tunisia; (ii) estimate the populations of Fsc and Fon in these fields based on pathogenicity tests of selected colonies; and (iii) assess the potential for development of Fusarium wilt and crown and root rot diseases in these fields based on soil bioassays using seedlings of a susceptible watermelon cultivar.

MATERIALS AND METHODS

Field survey Forty-one commercial watermelon fields from the main watermelon pro- duction areas of Tunisia were surveyed from 2000 to 2001 to study Fusarium wilt and Fusarium crown and root rot diseases. Disease incidence in each field was estimated, and the cultivars grown and crop history were recorded. This showed that cv. 'Charleston Gray' was the most commonly cropped in the north and south, whereas 'Crimson Sweet' was dominant in the center of Tunisia. A visual estimation of disease incidence, which was independent of the latest culture in the field, was absent in a few fields but extremely severe in others (Table 1).

Soil sampling and processing To quantify the inoculum density of Fusarium spp., 20 soil samples were collected from each field. Soil samples were taken at harvest, around symptomatic plants, following a "W" sampling pattem (27). Each soil sample (,,~500 g of soil) was collected from a depth of 10-20 cm and at a distance of 25 cm from a plant. All soil samples were stored at 4~ until use. Soil samples from the same field were combined into one sample, mixed thoroughly, and kept in the laboratory to be air-dried for 15-20 days. The population of Fusarium spp. in each soil sample was determined by plating aliquots of a soil dilution series. A 15-g sub-sample of soil was added to 100 ml of distilled sterilized water. The resulting soil suspension was mixed for 5 rain using a magnetic stirrer. One ml of this suspension was plated onto ten petri dishes containing Komada's medium (1 g K~HPO4; 0.5g KCI; 0.25g MgSO4.7H20; 2 ml FeEDTA; 2 g L-asparagine; 10 g galactose; 20 g agar; 0.5 g MgSO4.7H20; 0.1g CaCI2.2H20; 7.5 mg FeSO4.7H20; 2 mg

150 N. Boughalleb and M. El Mahjoub

MnSO4.7H20; 6 mg CuSO4.5H20; 75 mg ZnC12; 9 mg (NH4)6MoTO24; 5 #g biotine; 1 mg thiamine; 20 g agar; per liter of distilled water) (17). Three replicates of soil dilutions were prepared for each field sampled. All plates were incubated at 25~ for 4-5 days.

Identification of Fusarium spp. All colonies which showed cultural characteristics that were similar to Fusarium spp. were transferred to PDA (potato dextrose agar: 40 g l -x) and Spezieller N/ihrstoffarmer agar (SNA, composed of 1 g potassium di-hydrogen phosphate KH2PO4; 1 g potassium nitrate KNO3; 0.5 g magnesium sulfate MgSO4.7H20; 0.5 g potassium chloride KCI; 0.2 g glucose analar; 0.2 g sucrose analar; 20 g agar oxide no. 3; per liter of distilled water). The macroscopic features were determined on PDA adjusted to pH 6.5-7, whereas the microscopic determination was done on SNA (26). Cultures on PDA and SNA were incubated at 22-25~ 12:12 L:D, for 4 days. The diameter and coloration of the colonies and the aspect of the mycelia were noted. The microscopic features were examined after 3 additional days of incubation. Colonies were identified as E solani or E oxysporum according to morphological criteria indicated by Booth (2) and Nelson et al. (23). The total number of colonies identified as E solani or E oxysporum was counted for each soil sample. The isolates were stored on PDA in tubes maintained at 5~ or in glycerol 50% at-20~ until use.

Pathogenicity to watermelon seedlings The ratio of pathogenic to non-pathogenic colonies of E solani and E oxysporum for each field sampled was determined by pathogenicity tests of random, representative colonies using susceptible watermelon seedlings (cv. 'Giza'). The number of colonies selected for pathogenicity tests from each field was determined as follows: (i) all colonies were tested if the total number of colonies on all the plates for a field was <20; and (ii) 20 colonies were randomly selected if the total number of colonies was >20. Thus, for E solani, for all fields, we chose an arbitrary 20 colonies per field, since the total number per field was >20 colonies. However, for E oxysporum, for approximately 17 fields, we used all colonies (Table 2).

The pathogenicity of all selected isolates was assessed on watermelon seedlings of the susceptible cv. Giza. Spore suspensions were prepared from cultures which originated from a single selected colony, grown on potato dextrose broth (PDB), using a rotary shaker at room temperature (25~ for 14 days and adjusted to 106 conidia m1-1. The seeds were surface- disinfected in 5% sodium hypochlorite solution for 5 min, rinsed with sterilized water, and sown on vermiculite to germinate. When the first true leaf was evident (,-~2 weeks after planting), the seedlings were uprooted and the roots were washed under tap water. The seedlings were root-dipped in the inoculum suspension for 15-20 seconds, swirled several times and transplanted into 7.5-cm-diam pots, with three seedlings per pot, containing vermiculite; and five pots per isolate, for a total of 15 seedlings per isolate. Controls were prepared by root-dipping the seedlings in sterile distilled water (18). All plants were maintained in the greenhouse and fertilized regularly once a week. The average air and soil temperatures during the experiment were 27 ~ and 24.7~ respectively. Evaluation of disease in the seedlings and assessment of pathogenicity were carried out 1 4-21 days after inoculation.

Disease potential of surveyed fields The potential for development of Fusarium wilt or Fusarium crown and root rot in each of surveyed watermelon fields was evaluated by sowing seeds of watermelon cv. Giza in 216 cm 3 pots filled with 170 cm 3 of soil collected from each field (30). Each field sample consisted of three pots, which served as three

Phytoparasitica 34:2, 2006 151

replicates, each with ten seedlings. A loamy sand soil that had been steamed to be free of Fusarium spp. served as the non-infested control soil. All plants were maintained in the greenhouse as described above. The incidence of infected seedlings was assessed weekly for a period of 6 weeks after planting. Colonies isolated from infected seedlings onto Komada's medium were identified on PDA and SNA as described above.

Data analysis The relations between percentage of infected plants (disease incidence) and inoculum density in soils was determined by regression analysis. Data were analyzed using the software SPSS program (SPSS Inc. Headquarters, Chicago, IL, USA).

RESULTS

Fusarium isolation and identification Different types of Fusarium colonies developed on Komada medium. Colonies of E solani and E oxysporum differed from other Fusarium species in color and morphology and were confirmed by microscope identification. Their growth on PDA was rapid, with abundant aerial mycelium. The surface of the colonies was covered with sporodochia, of cream or blue color. The growth on SNA showed that microconidia were formed in false heads on long branched monophialides (F. solani) or on short branched monophialides (E oxysporum) and in the presence of abundant chlamydospores (4,23). The numbc~ ol ,. solani colonies that were recovered from the field soils was higher than the number of E oxysporum colonies (Table 2), the total number of colonies being, respectively, 20,078 and 1434 CFU g-1 soil (Table 2).

Pathogenicity tests on watermelon seedlings Identification of pathogenic isolates of Fsc and Fon was done according to the symptoms observed: crown and root rot for Fsc, and wilt for Fon. Most of the selected E solani colonies were pathogenic to watermelon seedlings and caused crown and root rot and were subsequently identified as Fsc. Only a few of the F. oxysporum isolates that were recovered from the soil samples and found to be pathogenic to watermelon seedlings were identified as Fon, responsible for vascular wilt of seedlings. The symptoms caused by Fsc appeared when plants approached maturity, and exhibited a severe conical rot at the base of the stem and the upper portion of the taproot, causing yellowing and wilting of the leaves. Within a few days of the first symptoms of crown rot, diseased plants usually died. Fon penetrates into the plant through the root system and the pathogen quickly invades the lymphatic vessels (xylem), which is the cause of its collapse. As a consequence, the plant cannot absorb water, leading to the wilt of some branches or even the entire plant. The lymph vessels turn brown and smell like rotten almonds. It is important to note that there was no root galling on the collected plants. The number of pathogenic isolates of the two pathogens is shown in Table 2. These fungi were re-isolated, confirming Koch's postulate. The total number of Fsc and Fon colonies detected were, respectively, 17,607.6 and 1185.9 CFU g-1 soil (Table 2).

Inoculum density of E solani and E oxysporum in soil A large range in inoculum density was observed for E solani (Table 2). In most of the fields (87%), the inoculum density varied between I 114-98 and 11414-117 CFU g-1 soil. Three fields showed a higher level of inoculum density, varying from 1309+35 to 1600+191 CFU g-1 soil. In a few surveyed fields (5%), the inoculum density of F. solani was very low (365:31 to 89-t-29 CFU g-1 soil). F. oxysporum was isolated in most of the fields but with lower inoculum density than that of F. solani (Table 2). In most of the fields (73%), the inoculum density was between zero and 364-12 CFU g-1 soil. In others (15%), the inoculum density of F.

152 N. Boughalleb and M. El Mahjoub

TABLE 1. Crop history, watermelon cultivars and estimation of disease incidence in surveyed fields in various regions and locations in Tunisia

Region Location Field Preceding Survey date Cultivar grown Disease incidence z crop

North Oued Mliz F1 Cereal 5 July 01 Charleston Gray Low F2 Cereal 5 July 01 Charleston Gray Low

Testour F3 Solanaceae 3 July 01 Crimson sweet Extremely severe F4 Solanaceae 3 July 01 Charleston Gray Extremely severe

Gaafour F5 None 24 July 01 Crimson sweet Extremely severe F6 None 24 July 01 Charleston Gray Extremely severe F7 None 24 July 01 Charleston Gray Absent

Laaroussa F8 Watermelon 24 July 01 Crimson sweet Severe F9 Watermelon 24 July 01 Charleston Gray Extremely severe

Middle Chebika F10 None 6 June 01 Crimson sweet Very low Fll None 6 June 01 Crimson sweet Very low FI~ None 6 June 01 Crimson sweet Very low Fla None 6 June 01 Crimson sweet Very low

Souassi F14 None 11 July 01 Crimson sweet Low FI~ None 11 July 01 Crimson sweet Low

Chorben F16 None 11 July 01 Crimson sweet Very low F17 None 11 July 01 Crimson sweet Very low FlS None 11 July 01 Crimson sweet Very low F19 None 11 July 01 Crimson sweet Very low

Jelma F2o Watermelon 21 June 01 Crimson sweet Low F21 Watermelon 21 June 01 Crimson sweet Low F22 Watermelon 21 June 01 Crimson sweet Low

Ben Aoun F23 None 21 June 01 Crimson sweet Absent F24 Nonee 21 June 01 Crimson sweet Absent

Sebbala F25 Watermelon 21 June 01 Crimson sweet Low F26 Watermelon 21 June 01 Charleston Gray Low

Regueb F27 None 23 May 01 Crimson sweet Low F28 None 23 May 01 Crimson sweet Very low

Jebeniena F29 Watermelon 5 June 01 Crimson sweet Low Fao Watermelon 5 June 01 Crimson sweet Low Fal Watermelon 5 June 01 Crimson sweet Severe

South Skhira Fs2 Cereal 23 May 01 Crimson sweet Severe Fa3 Cereal 23 May 01 Charleston Gray Low Fa4 Cereal 23 May 01 Charleston Gray Low

Metouia F35 Muskmelon 13 June 01 C.S and Severe Ch.Gray

F36 Watermelon 13 June 01 Charleston Gray Extremely severe Gafsa F37 None 21 June 01 Charleston Gray Extremely severe

F38 None 21 June 01 Charleston Gray Extremely severe Medenine Fa9 None 14 June 01 Charleston Gray Absent

F4o None 14 June 01 Charleston Gray Absent F41 None 14 June 01 Charleston Gray Very low

ZDisease incidence was estimated visually.

oxysporum varied from 53~30 to 97~57 CFU g-1 soil, and in five fields it was more than

100 CFU g-1 soil. The highest inoculum density of E oxysporum was found at Gaafour

(Fs) (200+ 18 CFU g-a soil) (Table 2). The percentage of visually estimated symptoms in

the fields is related to the inoculum density of Fusarium spp. (Tables 1 and 2).

Fsc and Fon soil populations were estimated according to the results obtained in the

pathogenicity tests with selected colonies. Fsc inoculum varied between 9 and 1600 CFU

g-1 soil with the highest density at Laaroussa (1600 CFU g-1 soil). For Fon, it ranged

Phytoparasitica 34:2, 2006 153

TABLE 2. Inoculum densities (expressed as CFU g-1 soil) of Fusarium solani and F. oxysporum

in soils from watermelon fields in Tunisia, and estimation of inoculum densities of E solani f.sp.

cucurbitae (Fsc) and E oxysporum f.sp. niveum (Fon) and their disease potential assessed by soil

bioassays

Region Location Field Number of CFU g - 1 soil Disease incidence (%) F. solani z E Fsc v Fon crown and wilted

oxysporum root rot- seedlings affected seedlings

North Oued Mliz F1 5404-125 534-30 486 50.4 42 15 F2 7294-106 28 4-24 656 28 50 4

Testour F3 11414-117 1144-81 1084 114 58 28 F4 1309+35 1894-67 1244 56 59 16

Gaafour F5 10654-167 2004-18 1012 200 54 48 F~ 14094-242 1464-56 1268 132 62 32 Fr 5614-64 514-45 449 41 40 8

Laaroussa F8 16004-191 1044-20 1600 99 67 29 F9 9004-31 0+0 765 0 52 0

Middle Chebika Fro 1774-28.5 44-3.5 159 0 5 0 Fll 2574-70.8 24-2 218 0 12 0 F12 3424-45.5 234-23 342 18 23 0 F13 1824-102 34-3 127 0 5 0

Souassi Ft4 3844-131 54-1 211 0 41 0 F15 6244-85 44-2.7 468 0 58 0

Chorbene F16 1724-142 94-4.5 43 2 14 0 Fir 2594-52 04-0 220 0 20 0 F18 3454-123 254-16 293 2.5 26 0 F19 1844-44 04-0 119 0 10 0

Jelma F20 2424-50.7 04-0 169 0 23 0 F21 2114-37.7 624-47 200.5 62 20 13 F22 4624-71 7 4-3 416 1 33 0

Ben Aoun F23 1114-98 14-1 33 0 0 0 F24 364-31 04-0 9 0 0 0

Sebbala F25 4154-49 04-0 373.5 0 28 0 F26 2094-69 94-4.5 177.7 2 18 0

Regueb F27 6224-38.6 14-1 497.6 1 46 0 F28 5714-62 54-5 456.8 5 39 0

Jebeniena F29 5124-160 584-27 486 46.4 33 10 F3o 3234-66 974-57 290.7 97 20 25 Fal 7904-187 54-5 750.5 5 57 0

South Skhira F32 9734-120.5 184-13 973 18 80 0 F33 4474-61 544-41 357.6 54 35 10 F34 5794-267 364-12 434.3 36 40 2

Metouia F35 1844-82 154-13 128.8 15 14 0 F36 2204-111 364-32 198 32.4 20 1

Gafsa F37 2064-39 164-7 144.2 16 17 0 F38 3114-106 54-5 279.9 5 25 0

Medenine F39 1334-124 04-0 73.2 0 12 0 F40 834-29 174-5 33.2 16.2 0 0 F4t 2584-100 314-20 232.2 31 17 0

Total 20078 1434 17607.6 1185.9 ZMean inoculum density (4- standard deviation) estimated using dilution plating; the value for each replicate was the average number of Fusarium colonies on ten plates containing Komada medium. ~Estimation of inoculum density using pathogenicity test of colonies selected from each field.

154 N. Boughalleb and M. El Mahjoub

from zero to 200 CFU g-1 soil (the highest density at Gaafour). In the central region, the inoculum densities ranged from 9 to 497.6 CFU g-1 soil for Fsc and from zero to 97 CFU g- 1 soil for Fon. In the fields surveyed in southern Tunisia, inoculum densities varied from 33.2 to 973 CFU g-1 soil and from zero to 54 CFU g-1 soil for Fsc and Fon, respectively. In the northern fields, the inoculum densities ranged from 486 and 1600 CFU g-1 soil for Fsc and from zero to 200 CFU g-1 soil for Fon.

ra

q) "O ~a e-,

t~

0a

A

0 .9

0 8

0 3

0 6

0 .5

0 4

0 .3

0 .2

0.1

0 A v

Y = 0 . 0 0 0 5 X + 0 1699

R 2 = 0 .668

i ,O�9

. . . - �9 �9 � 9

: : - . -

. . , t , f

I1' II, 0

41,

0 4 0 0 8 0 0 1 2 0 0 1 6 0 0

I n o c u l u m d e n s i t y ( C F U / g s o i l )

Fig. 1. Disease incidence of crown and root rot caused by Fusarium solani f.sp. cucurbitae in watermelon seedlings planted in different soil samples collected from fields in Tunisia.

Disease potential of watermelon fields Disease potential was assessed by soil bioassays. The incidence of Fusarium wilt or Fusarium crown and root rot in cv. Giza seedlings planted in soil samples collected from the fields was determined for each soil sample (Table 2). Fusarium crown- and root-rot-diseased seedlings were observed in most of the soils (93%). A low percentage of infected seedlings (5-20%) was recorded in 34% of the soil samples; 27% of the samples had between 21% and 40% diseased plants; and in 24% of the soil samples, 41% to 60% of the seedlings were infected by Fsc. The highest levels of crown and root rots (62% and 80%, respectively) were observed in soil samples from Gaafour and Skhira (7% of fields). No diseased watermelon seedlings were found on soil samples from Ben Aoun, where Fsc inoculum density varied from 9 to 33 CFU g-1 soil, or from Medenine, where Fsc inoculum density was 16.2% (Table 2). Unlike the crown and root rot, Fusarium wilt was observed in only 34% of the soil samples. Twenty-two percent of soils showed a low percentage of wilted plants (1-16%). Only a few soils (10%) had a level of infected seedlings between 25% and 32%. A relatively high level of disease (48%) was observed among seedlings grown in soil from one field at Gaafour.

Regression analysis indicated a significant correlation (P<0.001) between the percent-

Phytoparasitica 34:2, 2006 155

o r u

0 .6

0.5

0.4

0.3

0.2

0.i

0

Y = 0.003 X - 0.014

"

0 50 100 150 2 0 0

Inoeulum density (CFU / g soil)

Fig. 2. Disease incidence of wilting caused by Fusarium oxysporum f.sp. niveum in watermelon seedlings planted in different soil samples collected from fields in Tunisia.

age of infected plants and the soil inoculum density for both Fsc and Fon (Figs. 1 and 2). A positive linear model described this relationship, which was characterized by the equation Y=0.000X+0.165, with a coefficient of determination (R 2) of 0.67 for Fsc, and Y=0.003xX-0.014 with R 2 of 0.88 for Fon, showing that a higher percentage of disease corresponded with a higher soil inoculum density. Based on these equations, the estimated inoculum densities required to cause 50% disease incidence (DIso) on cv. Giza were 670 CFU g- 1 soil and 171 CFU g- 1 soil for Fsc and Fon, respectively.

DISCUSSION

Inoculum densities of E solani and E oxysporum in watermelon fields located in production areas in Tunisia were determined. The study showed that inoculum of F. solani was more prevalent than that of E oxysporum, which was less or absent in some of the surveyed fields. Our results revealed that the inoculum densities of E solani and E oxysporum in Tunisian watermelon fields varied respectively from 36-4-31 to 1600-t-199 CFU g-1 soil and from zero to 200+ 18 CFU g-1 soil. Results obtained by Netzer (24) in Israel showed a range of E oxysporum from 400 to 1400 CFU g-1 soil in heavily infected fields cropped with watermelon cv. Sugar Baby. Hopkins and Elmstrom (15) noted that concentrations of F. oxysporum propagules were not very useful in monitoring Fusarium wilt pressure in soil, since saprophytic isolates of E oxysporum are ubiquitous in soil, and this would require pathogenicity tests of many E oxysporum isolates from each plot. They suggested a greenhouse bioassay to measure the disease pressure of the soil infested by E oxysporum populations.

Pathogenicity tests showed that most of the E solani and E oxysporum isolates were pathogenic to watermelon seedlings and were identified as Fsc and Fon, respectively. In the present study, Fon soil inoculum density was low, varying from zero to 200• CFU

156 N. Boughalleb and M. E1 Mahjoub

g-1 soil. In Maryland and Delaware watermelon fields, inoculum densities of Fon were higher, ranging from 100 to 1200 CFU g-1 soil (29). This research is the first to determine inoculum density of E so lan i and Fsc in watermelon fields showing symptoms of Fusarium crown and root rot disease. According to our data, F u s a r i u m populations in the soils of most watermelon fields were composed predominantly of Fsc.

Assessments of the potential development of Fusarium wilt and Fusarium crown and root rot diseases in these fields were based on assays using seedlings of a susceptible watermelon cultivar, sown in soil samples from infested fields. These assays showed that the incidence of crown and root rot disease and Fusarium wilt disease of watermelon was significantly correlated with inoculum density of Fsc and Fon (R2=0.67 and 0.88, respectively). Our findings are in agreement with the results obtained by Zhou and Everts (29), who showed that Fusarium wilt of watermelon was significantly correlated with inoculum density of Fon and the estimated inoculum density required to cause DIso on Sugar Baby, which was 367 CFU g-1 soil. However, we found that inocutum density causing 50% incidence on cv. Giza reached 171 CFUg -1 soil. This difference could be due to cultivar, climatic conditions and edaphic factors. Furthermore, the inoculum density in the surveyed watermelon fields may have been over- or underestimated because soilborne fungi are not uniformly distributed in infested fields and perhaps also because of the methods used, like air-drying the soil - which might cause degradation of the pathogen population.

To the best of our knowledge, this report is the first on the relationship between inoculum density of Fsc and disease incidence in watermelon seedlings. The information presented here could be very useful, since knowledge about the range of inoculum densities of F u s a r i u m spp. in commercial fields would help us to employ multiple tactics for Fusarium wilt management; and to determine inoculum concentrations of these pathogens needed to infest soils for research purposes in greenhouse or field trials. Moreover, it is possible to adopt the employed equation to estimate the disease pressure in fields where only one or both pathogens are present. This correlation between disease incidence and inoculum density could vary if there are only one or two pathogens in the soil sample.

ACKNOWLEDGMENTS

The authors sincerely appreciate the suggestions for improvement of the manuscript made by Dr. J. Armengol, Instituto Agroforestal Mediterr,'ineo, Universidad Polit6cnica de Valencia, Valencia, Spain.

REFERENCES

1. Armengol, J, Jos6, C.M., Moya, M.J., Sales, R., Vicent, A. and Garcia-Jim6nez, J. (2000) Fusarium solani f.sp. cucurbitae race 1, a potential pathogen of grafting watermelon production in Spain. OEPP Bull. 30:179- 183.

2. Booth, C. (1971) The Genus Fusarium. Commonwealth Mycological Institute, Kew, Surrey, UK. 3. Boughalleb, N., Armengol, J. and El Mahjoub, M. (2005) Detection of races 1 and 2 of Fusarium solani f.sp.

cucurbitae and their distribution in watermelon fields in Tunisia. J. Phytopathol. 153:162-168. 4. Boughalleb, N. and El Mahjoub, M. (2005) D6tection des races 0, 1 et 2 de Fusarium oxysporum f.sp. niveum

et leur distribution dans les r6gions de production de la past6que en Tunisie. Bull. OEPP 35:253-260. 5. Bruton, B.D. and Damicone, J.P. (1999) Fusarium wilt of watermelon: Impact of race 2 of Fusarium

oxysporum f.sp. niveum on watermelon production in Texas and Oklahoma. Subtrop. Plant Sci. 51:4-9. 6. Bruton, B.D., Patterson, C.L. and Martyn, R.D. (1988) Fusarium wilt (E oxysporum f.sp. niveum race 2) of

watermelon in Oklahoma. Plant Dis. 72:734 (abstr.). 7. Cirulli, M. (1972) Variation of pathogenicity in Fusarium oxysporum f.sp. niveum and resistance in

watermelon cultivars. Actas 111 Congr. Uniao Fitopatol. Mediterr. pp. 491-500.

Phytoparasitica 34:2, 2006 157

8. Doidge, E.M. (1938) Some South African Fusaria. Bothalia 3:331-483. 9. Elmstrom, G.W. and Hopkins, D.L. (1981) Resistance of watermelon cultivars to Fusarium wilt. Plant Dis.

65:825-827. 10. Fantino, M.G., Fantuz, E and Gennari, S. (1989) Study of squash rot caused by Fusarium solani f.sp.

cucurbitae. Dif. Piante 12:147-154. 11. Garcia-Jimenez, J., Armengol, J., Moya, M. and Sales, R. (1997) First report of Fusarium solani f.sp.

cucurbitae race 1 in Spain. Plant Dis. 81:1216 (abstr.). 12. Gonzalez-Torres, R., Melero-Vara, J., Gomez-Vazquez, J. and Jimenez Diaz, R.M. (1993) The effects of soil

solarization and soil fumigation on Fusarium wilt of watermelon grown in plastic houses in south-eastern Spain. Plant Pathol. 42: 858-864.

13. Hall, D.H., Gubler, W.D. and Sciaroni, R.H. (1981) Fusarium fruit rot of cucurbits. Calif. Plant Pathol. 54:3 (abstr.).

14. Hawthorne, B.T., Rees-George, J. and Broadhurst, P.G. (1992) Mating behaviour and pathogenicity of New Zealand isolates of Nectria haematococca (Fusarium solani). N.Z. J. Crop Hortic. Sci. 20:51-57.

15. Hopkins, D.L. and Elmstrom, G.W. (1984) Fusarium wilt in watermelon cultivars grown in a 4-year monoculture. Plant Dis. 68:129-131.

16. Ioannou, N. and Poullis, C.A. (1991) Fusarium wilt of resistant watermelon cultivars associated with a highly virulent local strain of Fusarium oxysporum f.sp. niveum. Tech. Bull. Agric. Res. Inst. Nicosia, Cyprus no. 129.

17. Komada, H. (1975) Development of a selective medium for quantitative isolation of Fusarium oxysporum from natural soil. Rev. Plant Prot. Res. 8:115-125.

18. Latin, R.X. and Snell, S.J. (1986) Comparison of methods for inoculation of muskmelon with Fusarium oxysporum f.sp. melonis. Plant Dis. 70:297-300.

19. Martyn, R.D. (1987) Fusarium oxysporum f.sp. niveum race 2: A highly aggressive race new to the United States. Plant Dis. 71:233-236.

20. Martyn, R.D. and Bruton, B.D. (1989) An initial survey of the United States for races of Fusarium oxysporum f.sp. niveum. HortScience 24:696-698.

21. Messiaen, C.M., Blancard, D., Rouxel, E and Lafou, R. (1991) Les Maladies des Cultures Maraich~res. 3~me 6d. INRA, Paris, France.

22. Nagao, H., Sato, K. and Ogiwara, S. (1994) Susceptibility of Cucurbita spp. to the cucurbit root-rot fungus, Fusarium solani f.sp. cucurbitae race 1. Agronomie 2:95-102.

23. Nelson, P.E., Toussoun, T.A. and Marasas, W.EO. (1983) Fusarium Species: An Illustrated Manual for Identification. Pennsylvania State University Press, University Park, PA, USA.

24. Netzer, D. (1976) Physiological races and soil population level of Fusarium wilt of watermelon. Phytopara- sitica 4:131-136.

25. Sherf, A.F. and MacNab, A.A. (1986) Vegetable Diseases and Their Control. 2 "``/ed. Wiley, New York, NY. 26. Summerell, B.A., Salleh, B. and Leslie, J.E (2003) A utilitarian approach to Fusarium identification. Plant

Dis. 87:117-128. 27. Wuster, G. (1991) La mycologie en pathologie v6g6tale. Techniques de Laboratoire. Minist&e de

l'Agriculture et des Ressources Hydrauliques de Tunisie. Service de la Protection des V6g6taux. Rapport de la Mission de Coop6ration Technique, Tunisie.

28. Zhou, X.G. and Everts, K.L. (2001) First report of the occurrence of Fusarium oxysporum f.sp. niveum race 2 in commercial watermelon production areas of Maryland and Delaware. Plant Dis. 85:1291 (abstr.).

29. Zhou, X.G. and Everts, K.L. (2003) Races and inoculum density of Fusarium oxysporum f.sp. niveum in commercial watermelon fields in Maryland and Delaware. Plant Dis. 87:692-698.

30. Zh•u• X.G. and Everts• K.L. (2••4) Quantificati•n •f roots and stem c•••nizati•n •f waterme••n by Fusarium and its use in evaluating resistance. Phytopathology 94:832-841.

158 N. Boughalleb and M. El Mahjoub