J. Phytopathology 152, 514–518 (2004) 2004 Blackwell Verlag, Berlin ISSN 0931-1785
Biological Control of Heterodera cruciferae (Tylenchida: Heteroderidae) Franklin 1945 with Fluorescent Pseudomonas spp. H. M. Aksoy and S. Mennan Authors address: University of 19 May, Faculty of Agriculture, Plant Protection Department, Samsun 55139, Turkey (correspondence to H. M. Aksoy. E-mail:
[email protected]) Received November 3, 2003; accepted July 11, 2004 Keywords: cyst nematode, fluorescent Pseudomonas, plant–parasitic nematode
Abstract This study was conducted to access the ability of fluorescent Pseudomonas spp. to suppress cyst nematode Heterodera cruciferae, which was caused by reproduction. The yield loss of cruciferous plants in infested soil with H. cruciferae was examined in in vitro and in vivo experiments. For this purpose, fluorescent Pseudomonas spp. was isolated from infested soil with cyst nematode H. cruciferae and a selected strain of fluorescent Pseudomonas [Fluorescent Pseudomonas strain no. 6 (FPs6)] used on cyst, females and eggs of H. cruciferae to study the biological control. In the in vitro study, eggs of H. cruciferae were infected by FPs6 on King’s B Agar and consequently the growth and hatching of eggs were inhibited. Furthermore, J1 and J2 being grown in diseased eggs as they were infected by FPs6. In the in vivo study, plant lengths, leaf areas, wet weights, dry weights and root lengths of seedlings were investigated. There were no considerable differences among FPs6, FsP6 + Cyst and Control groups on plant length and root length (P > 0.05). But there were considerable differences between Cyst and Control groups on plant length and root length (P > 0.05).
Introduction Plant–parasitic nematode Heterodera spp. are economically important pests in agriculture. Each species generally attacks a small number of plant species, often within one family, and damage is often economically important. Typical symptoms include chlorotic foliage, stunting and severe wilting as a result of abnormal water stress during hot weather. Root development may be severely stunted (Jensen, 1972). The body wall of female of Heterodera spp. generally encloses most of her eggs, though some may be extruded in a posterior egg sac. This wall hardens and dries to protect the eggs from adverse environmental conditions and predation. Nematodes hatch from eggs as second stage juveniles, which burrow into root tissue and form specialized feeding sites (syncytia). Females remain in U. S. Copyright Clearance Centre Code Statement:
this sedentary position and pass through two more stages to become adults, which lay scores of eggs internally. Following egg production, females die and their bodies harden to form protective structures around the eggs (cysts). The protective cyst make these pests more difficult to control by nematicides and cultural practices. Resistant cultivars are not always available and may produce lower yields than the susceptible cultivars. In the past 20 years three developments have occurred which have had significant developments on the studies and the prospects for the biological control of plant– parasitic nematodes. First, several nematicides have been withdrawn from the market in developed countries because of the use of chemicals for the control of these pests which is not economical on many crops. Breakdown products may be highly toxic to mammals, and have polluted groundwater and presumably other areas where these pesticides are widely used (Jensen, 1972; Thomason, 1987; Hay and Bateson, 1996). Therefore, alternative strategies for control, including the use of biological agents are being extensively researched by nematologists. Second, in many soils nematophagous fungi and bacteria increase under some perennial crops, and crops grown in monoculture. These organisms may control some nematode pests, including cyst and root-knot nematodes. Such nematode-suppressive soils have been reported from around the world and include some of the best documented cases of effective biological control of nematode pests (Kerry, 1988; Stirling, 1991). Finally, a number of commercial products based on nematophagous fungi and bacteria have been developed, but all so far have had only limited success. Some bacterial strains have the ability to multiply and spread in the rhizosphere to colonize sites on the root and to act by direct contact with parasites (Becker et al., 1988; Oostendorp and Sikora, 1989; Kloepper et al., 1992; Racke and Sikora, 1992; Kluepfel et al., 1993). While many reports relate the use of bacteria for biological control, only one reference to this type
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Biological Control of H. cruciferae
of control in Turkey was found. Fourteen nematode species were infected by the bacterium Pasteuria penetrans including, Tylenchulus semipenetrans, Meloidogyne incognita, Meloidogyne javanica, Meloidogyne arenaria, Pratylenchus penetrans, and Pratylenchus thornei, all important plant parasites (Eleks¸iog˘lu, 1995). Breakdown products of nematicides may be highly toxic to mammals and have polluted groundwater and presumably other areas where these pesticides are widely used. For this reason, the researchers focused on using biocontrol agents in place of chemical agents during recent years. The objectives of the current study were (a) to isolate fluorescent strains of bacteria Pseudomonas spp. from soil, and (b) to test them as potential biocontrol agents of the cyst nematode, Heterodera cruciferae.
Materials and Methods
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serial dilutions (103–104) were prepared. Sample dilutions were placed on King’s B Agar (KBA) (King et al., 1954) and were incubated at 24–26C for 24–48 h. In the second method, abnormal-shaped cysts were separated from normal cysts using a stereo-binocular microscope. Approximately, 100 abnormal-shaped cysts for each sample were used for extraction and examination. These cysts were surfacesterilized in 0.5% sodium hypochlorite (NaOCl), rinsed several times in sterile water, gently blotted dry on paper towels, placed on KBA and incubated at 24–26C for 24–48 h. Bacterial colonies were examined under a fluorescent light with a long wavelength (366 nm) ultraviolet lamp is used for identification of fluorescent strains and further identification was based on colony morphology and fluorescent character was according to standard diagnostic methods (Lelliott and Stead, 1987; Kiewnick and Sands, 2001).
Soil sampling
Sixty cabbage fields were investigated in Northern Black Sea region during 2001–2003. In each field, approximately 20 soil cores were taken from the cabbage root zones with a soil probe to a depth of 15–25 cm in a zigzag pattern in an area of approximately 5 ha. The 20 soil cores from a field were mixed and considered one soil sample per field (Chen and Chen, 2002). The samples were stored in plastic bags at 4C and processed within a week. Extraction of cysts and collecting of eggs
Cyst and females were extracted from soil by suspending the soil in water and then by through pouring. For this purpose, soil samples were washed through the upper 840 l aperture (20-mesh) sieve and cysts were collected on the 250 lm aperture (60-mesh) sieve. Contents of this sieve were rinsed thoroughly and cysts were collected in a disk. An aspirator was used to remove cysts from wet debris (Shepherd, 1987). Cyst and second stage juveniles were measured for identification (Mulvey and Golden, 1983). The glycerine–agar technique was used for vulval cone preparations of cyst (Correia and Abrantes, 1997). All cysts were identified as H. cruciferae. Approximately 100 cysts for each sample were used for extraction and examination of eggs. Eggs were released by breaking each cyst into two pieces using a surgical knife sterilized with 70% ethanol and were collected with a slim brush. Eggs that appeared to be infected with bacteria were separated, counted and identified at species level and stained by adding a few drops of Lacto-phenol. Abnormalshaped cysts were separated from normal cysts using a stereo-binocular microscope (100 and 200· magnification). Bacterial isolations
Two methods were used to isolate. In the first method, each soil sample was sieved through a 1-mm mesh sieve, mixed at a ratio of 1 : 10 with sterile distilled water and shaken thoroughly for 60 min and then
Treatment of cyst with bacteria
The study was set up according to four replications and repeated two times concurrently. Normal-shaped cysts were surface-sterilized in 0.5% NaOCl, rinsed several times in sterile water and gently blotted dry on paper towels. The eggs were released from these cysts by breaking into two pieces by surgical knife sterilized with 70% ethanol and were collected with a slim brush. Each Petri dish containing bacterial strain no. 6 grown on KBA at 24–26C for 24–48 h was seeded with 10 eggs. After 12, 24, 48, 96, 240 and 480 h, both diseased and normal eggs were placed in lacto-phenol and counted with the aid of a light microscope at 100– 200· magnification. The egg–parasitic index (EPI) was used to indicate bacterial parasitism of eggs in cysts or females in the Petri dish experiment (Chen and Chen, 2002). The EPI was recorded based on the following 0–10 scale: 0 ¼ no eggs colonized; 1 ¼ 1–10%; 2 ¼ 11–20%; 3 ¼ 21–30%; 4 ¼ 31–40%; 5 ¼ 41–50%; 6 ¼ 51–60%; 7 ¼ 61–70%; 8 ¼ 71–80%; 9 ¼ 81–90% and 10 ¼ 91–100% of eggs colonized. Treatment of seedlings with bacteria
Sandy-loam soils (sand 80%, clay 10% and silt 10%) used for seedling experiments were sterilized in autoclave at 121C for 20 min. Then, cabbage seeds sterilized with NaOCl 5% were planted in these soils. After 4–5 weeks, cabbages seedling roots were washed free of soil. In the pot experiments plant lengths, leaf areas, wet weights, dry weights and root lengths of seedlings were investigated and used for four treatments [Bacterial Strain (BS), Cyst (C), Bacterial Strain + Cyst (BSC) and Control (CO)]. Lateral roots of seedlings were cut with a scalpel at 3 cm deep in each pot experiment. Afterwards, roots of seedlings were soaked in 50 ml bacterial suspension containing 106–108 CFU/ml for 2 h in the BS treatment. Ten healthy cysts were applied to the root zone of seedlings in each pot in the C treatment. Roots of seedlings were soaked in 50-ml bacterial suspension containing 106–108 CFU/ml
Aksoy and Mennan
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Fig. 1 (a) Eggs of Heterodera cruciferae infected by fluorescent Pseudomonas strain no. 6 (FPs6). (b) Growth and hatching of eggs were inhibited by FPs6
Fig. 2 (a–c) Diseased eggs and J2 of Heterodera cruciferae: first stages (a), second stages (b), last stages of FPs6 infection (c)
for 2 h and 10 cysts were applied to the root zone of seedlings in each pot in the BSC treatment. Each treatments was repeated four times and three seedlings were planted to those containing no cysts and bacterial inoculum in the CO treatment. Experiments were conducted concurrently and terminated 80 days after inoculations. Pots were put into growing chamber at 25 ± 1C, relative humidity 70%, day light 16 h and 8 h dark conditions randomly and watered daily.
these only Cap no. 6 [Fluorescent Pseudomonas strain no. 6 (FPs6)], isolated from the root zone of cabbage, was selected for use in the Petri dish and pot experiments. Effect of fluorescent Pseudomonas strain on eggs of H. cruciferae
Statistical analyses
Eggs of H. cruciferae were infected by FPs6, and growth and hatching of eggs were inhibited (Fig. 1). Furthermore, J1 and J2 inside the eggs hatched as diseased juveniles infected by FPs6 (Fig. 2). The Control
The one-way anova of the spss program for Windows (SPSS Inc., Chicago, USA) was used and in all cases treatment N ¼ 4, plant observation n ¼ 4 and the significant probability level was 0.05 in average.
Table 1 Percentage of eggs of Heterodera cruciferae infected with fluorescent Pseudomonas strain no. 6 (FPs6) and egg–parasitic index (EPI)
Results Identification of fluorescent Pseudomonas strains
Six fluorescent strains were isolated from soil and cyst samples, and were identified as Pseudomonas spp. Three of these strains were isolated from soil and the other three strains from cyst samples. Colonies of Pseudomonas fluorescens were whitish, grey, raised with diffusible yellowish-green pigment that fluorescence blue under ultraviolet light (366 nm). Of
Time (h) 12 24 48 96 240 480
FPs6 rates (%)
EPI*
13.79 14.46 17.39 61.96 72.83 77.39
2 2 2 7 8 8
*EPI in cysts from which the bacteria were isolated. EPI scale: 0 ¼ 0%; 1 ¼ 1–10%; 2 ¼ 11–20%; 3 ¼ 21–30%; 4 ¼ 31–40%; up to 10 ¼ 91–100% eggs colonized.
Biological Control of H. cruciferae Table 2 Effects of treatment of cabbage seedlings with fluorescent Pseudomonas strain no. 6 (FPs6) on plant growth
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Treatment
N
FPs6 Cyst FPs6 + cyst Control
4 4 4 4
Plant height (mean ± SD) 38.000 36.333 39.667 42.333
± ± ± ±
3.20ab 1.15a 0.57ab 4.61b
Wet weight (mean ± SD) 13.887 11.536 16.233 12.663
± ± ± ±
2.04a 1.52a 1.11a 3.75a
Dry weight (mean ± SD) 3.113 1.973 2.420 2.800
± ± ± ±
1.39a 0.86a 1.05a 1.22a
Root length (mean ± SD) 17.00 8.67 16.67 16.33
± ± ± ±
1.73a 1.52b 1.52a 1.15a
Within the columns values with the same superscript letter are not significantly different (P > 0.05).
group normal J2s hatched after 48 h and they observed at each time interval. Levels of infected eggs with FPs6 ranged from 13.79 to 77.39% and EPI values ranged from 2 to 8% (Table 1). While percentages of females colonized by FPs6 were low, with values of 13.79, 14.46 and 17.39% after 12, 24 and 48 h, respectively, the corresponding values for 96, 240 and 480 h (61.96, 72.83 and 77.39%) were high.
The effect of FPs6 against H. cruciferae stems from hydrolytic enzymes such as chitinases and b-1,3 gluconases (Ramamoorthy et al., 2001) and antibiotics (Mahaffee and Kloepper, 1994) produced by pseudomonades. Also our results were not inharmonious with Oostendorp and Sikora (1990). Further experiments will assess the effectiveness of fluorescent Pseudomonas at reduced inoculum density, in combination with egg–parasitic bacteria over a long-time openfield experiments.
Effect of fluorescent Pseudomonas strain on seedlings
Differences were not large among BS, C and BSC treatments, and there were not large differences among BS, BSC and CO treatments (P > 0.05) (Table 2). There were considerable differences in plant length of C and CO treatments (P > 0.05). Differences among the treatments in terms of wet weight and dry weight were not large (P > 0.05). In contrast, differences between the C treatment and the other treatments were considerable (P > 0.05). Differences were negligible among the BS, BSC and CO treatments in terms of root length (P > 0.05).
Discussion Some studies have indicated antagonistic relationships between plant parasitic nematodes and bacterial pathogens, for example, Ditylenchus spp. suppressed carnation wilt caused by Pseudomonas caryophylii (Stewart and Schindler, 1956). In addition some of the bacteria called growth promoting rhizobacteria (PGPR) have been used against nematode pests as plant growth promoters (P. fluorescens, P. putida and P. gladioli) (Mahaffee and Kloepper, 1994), systemic resistance inducer (P. fluorescens) (Sikora, 1988, 1992; Oostendorp and Sikora, 1989, 1990; Sikora and HoffmannHergarten, 1992), early root inhibitors of Heterodera schachtii (Oostendorp and Sikora, 1989, 1990) and biological control agents of H. schachtii (Oostendorp and Sikora, 1989), H. glycines (Kloepper et al., 1992) and Globodera pallida (Racke and Sikora, 1992). In this study hatching rates of eggs decreased gradually with increasing time (24, 48, 96, 240 and 480 h) and J2s were diseased when they hatched. In pot experiment there were considerable differences between C and the other treatments (P > 0.05). There were negligible differences among the BS, BSC and CO treatments on root length (P > 0.05). This indicated that reducing the number of cysts may be at least partly compensated for an increase in inspected condition.
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