Multisite survey of soil interactions with infestation of root-knot nematodes (Meloidogyne spp.) by Pasteuria penetrans

Soil Biology & Biochemistry 34 (2002) 1417–1424 www.elsevier.com/locate/soilbio

Multisite survey of soil interactions with infestation of root-knot nematodes (Meloidogyne spp.) by Pasteuria penetrans Thierry Mateillea,*, David L. Trudgillb, Carmen Trivinoc, George Balad, Abdoussalam Sawadogoe, Effie Vouyoukalouf a

UMR IRD/CBGP, Campus International de Baillarguet, CS 30016, 34988 Montferrier-sur-Lez Cedex, France b SCRI, Invergowrie, Dundee DD2 5DA, UK c INIAP, Boliche Experiment Station, Box 7069, Guayaquil, Ecuador d CARDI, Central Experimental Station, St Augustine, Trinidad and Tobago, WI e INERA/DPV, B.P. 403, Bobo-Dioulasso, Burkina Faso f NARF/ISPOT, Agrokipio, Chania, Greece Received 9 April 2001; received in revised form 3 April 2002; accepted 15 May 2002

Abstract Differences in rates of infestation of root-knot nematode (Meloidogyne spp.) populations by the endospore-forming bacterium Pasteuria penetrans were not entirely due to inter/intra-specific variability of the organisms. Soil conditions, especially texture and chemical characteristics, are also involved. Focusing studies on vegetable crops, interactions between the occurrence of Meloidogyne spp. juveniles encumbered with spores of P. penetrans and some physico-chemical soil characteristics were analysed both between regions (West Africa, e.g. Burkina Faso and Senegal; South America, e.g. Ecuador; the Caribbean, e.g. Trinidad and Tobago; and Mediterranean Europe, e.g. Crete) and within Ecuador, Burkina Faso and Senegal. In Ecuador (clay or silty-clay soils), the mean proportion of infested juveniles was high (41.9%). In the sandiest soils, as in Senegal, there were very few infested juveniles (4.7%). In Crete, Burkina Faso, and Trinidad and Tobago, where the soils are siltier, the mean proportions of infested juveniles range between 14 and 24.4%. Multivariate analysis performed on the data from Senegal and Burkina Faso revealed that a significant increase of the mean clay content (1.4 and 10.5%, respectively) improved the mean proportions of infested juveniles (from 1.6 to 42.2% and from 10.7 to 79.4%, respectively). Influences of the soil texture and structure on the availability of the spores of P. penetrans to infest the juveniles of Meloidogyne spp. are discussed. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Bacteria; Meloidogyne spp; Nematodes; Pasteuria penetrans; Soil characteristics

1. Introduction The potential of Pasteuria penetrans (Thorne, 1940; Sayre and Starr, 1988) as a biological agent to control rootknot nematodes (Meloidogyne spp.) has been tested on various annual crops (Brown et al., 1985; Channer and Gowen, 1988; Daudi et al., 1990; Oostendorp et al., 1991; Zaki and Maqbool, 1992) and perennials (Stirling, 1984; Verdejo-Lucas, 1992). This organism produces dome shaped spores (3 –4 mm diameter) which lie free in the soil and adhere specifically to second stage Meloidogyne juveniles as they migrate in search of plant roots. Spores of P. penetrans have been described attached to more than 200 * Corresponding author. Tel.: þ 33-4-99-62-33-13; fax: þ33-4-99-62-3345. E-mail address: [email protected] (T. Mateille).

nematode species belonging to more than 90 genera. However, the greatest numbers of reports relate to P. penetrans attacking Meloidogyne spp. and these isolates show specificity for the genus Meloidogyne (Oostendorp et al., 1990). Specificity for one Meloidogyne species have been demonstrated (Stirling, 1985) as well as intra-specific (Bird et al., 1990). P. penetrans is an ubiquitous parasite of the genus Meloidogyne. It has been described from temperate regions in Europe, North America, North and South Africa and the Middle East, and even from pre-polar regions such as Iceland and Norway (Sayre and Starr, 1988). However, both P. penetrans and Meloidogyne spp. are most abundant in the tropical and the subtropical regions of Central America, West, East and Central Africa, and SouthEast Asia, in islands such as Caribbeans, New Caledonia and the Azores (Sayre and Starr, 1988, Que´ne´herve´, pers.

0038-0717/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 0 2 ) 0 0 0 8 5 - 8

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Table 1 Number of samples taken from vegetable crops of five countries and corresponding number of samples with Meloidogyne spp. or P. penetrans Number of samples

Burkina Faso

Crete

Ecuador

Senegal

Trinidad and Tobago

Total Crops Cabbage Celery Cucumber Eggplant Green pepper Lettuce Melon Okra Pea Pepper Potato Sweet potato Tomato Watermelon Other

130

22

207

200

73

4

13

1

3

2

10 4 10

67

17 7 17 2 9 19

10

166 5 2

15 13 84 11 28

7 21

3 1

63

18

With Meloidogyne spp.

69

22

205

178

29

With P. penetrans

44

5

67

53

16

comm.). Not all populations of Meloidogyne spp. are infested by P. penetrans, and in those populations that are, its abundance is very variable. This may partially explain the variable results obtained in agronomic trials where P. penetrans was used in an attempt to control Meloidogyne spp. The variability in the occurrence and abundance of P. penetrans are thought to due partly to inter- and intraspecific variability of Meloidogyne spp. and P. penetrans and to the susceptibility of P. penetrans (Brown and Smart, 1985; Stirling, 1985; Bird et al., 1990), and of its parasitism to soil conditions. Spores of P. penetrans can survive for many years in dry soils (Stirling et al., 1990). Spore attachment to Meloidogyne juveniles during their migration through the soil depends on the mobility of the juveniles (Hatz and Dickson, 1992) and is influenced by soil moisture (Brown and Smart, 1984; Oostendorp et al., 1990) and temperature (Stirling et al., 1986). Techniques for controlling plant-parasitic nematodes with biological agents are still being developed, but this ability to maintain nematode populations below an acceptable economical threshold is very irregular and varies with many factors. The lack of detailed knowledge regarding plant –soil interactions and the natural environment of plant-parasitic nematodes and of their natural enemies, are major factors limiting our effective use of biological agents. Consequently, the preservation or the re-establishment of biologically based equilibrium whereby damage by nematodes is prevented needs the promotion of determinist research on biological and physico-chemical interactive soil processes which affect the development of nematodes and their natural enemies. In each country involved in this study which has a

problem with Meloidogyne in vegetable crops, a series of structured surveys were made to characterise the species present and their importance, and the occurrence of P. penetrans. In these surveys, some of the soil physicochemical characteristics were analysed and correlated with the occurrence of Meloidogyne spp. juveniles infested by P. penetrans. Their distributions were compared between and within the countries in order to understand the influence of the environmental factors on the efficiency of the bacterium as a biocontrol agent.

2. Materials and methods 2.1. Sampling methods A wide range of vegetable crops were surveyed in Burkina Faso, Crete, Ecuador, Senegal and Trinidad and Tobago (Table 1). The number of fields sampled in each country varied according to the accessibility of the vegetable producing areas. In Burkina Faso, 32 sites were randomly sampled all over the country. In Crete, the samples were taken from near the cities of Chania, Falasarma, Koudoura, Plakias and Tibaki. In Ecuador, most of the samples were taken from all over the coast region (62 sites), and less in the Highlands (35 sites) and in the oriental Amazonia (11 sites). One site was sampled in the Galapagos. In Senegal, the most important producing areas were surveyed: 10 sites in the coast region (Niayes), 10 sites in the Sereer region, and eight sites in the Senegal valley. Usually, 10 soil cores (10 –20 cm deep in the rhizosphere) were obtained across each field along an M shaped path and combined to make one sample per field,

T. Mateille et al. / Soil Biology & Biochemistry 34 (2002) 1417–1424 Table 2 Soil factors analysed and corresponding labels used in the multivariate analysis Variable

Label

Variable

Label

Clay Fine silts Coarse silts Fine sands Coarse sands

Cla FSi CSi FSa CSa

Wilting point 4 pH EC Conductivity

Wp4 pH EC Con

Aluminium Calcium Carbonate Magnesium Potassium Sodium Sulfate

Al Ca HCO3 Mg K Na SO4

Carbon Nitrogen Meloidogyne in soil

C N Me

except in Senegal where they were sampled along the field transect. 2.2. Nematode extraction and P. penetrans detection The methods used to extract nematodes, especially Meloidogyne second stage juveniles (J2s), from the soil varied according to the standard techniques available in each country. In Burkina Faso and Senegal, elutriation methods (Seinhorst, 1962) were used. In Ecuador and Trinidad and Tobago, J2s were extracted using the Baermann tray technique (Southey, 1986). In Crete, the nematodes were extracted by decanting and sieving (Barker, 1985). In all laboratories, the Meloidogyne J2s were counted in plastic counting dishes and the numbers determined per dm3 of soil. Twenty nematode juveniles were randomly observed under an inverted microscope and the percentage encumbered with spores of P. penetrans was estimated. 2.3. Soil analysis Soil analyses were done on those soils with P. penetrans to determine the proportion of clay (0 –2 mm), fine silt (2 – 20 mm), coarse silt (20 –50 mm), fine sand (50 – 200 mm), coarse sand (200 –2000 mm) particles and organic matter. The soil moisture (%) corresponding to the permanent wilting point 4.2 (Wp4), the pH H2O, the cation exchange capacity (EC), the electrical conductivity (free ions) and the concentrations in phosphorus, exchanged calcium, magnesium, sodium and potassium were also determined. 2.4. Statistical analysis All the soil variables were labelled as it is listed in Table 2. A principal component analysis (PCA) was applied on all the samples in each country. This PCA gave a loading plot for the soil variables defined by the two most significant eigenvalues (PC1 and PC2), and a score plot for the samples.

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For Ecuador, Burkina Faso and Senegal (where more than 100 samples were taken from each), proportion classes of infestation were established according to the percentage of juveniles of Meloidogyne spp. infested with P. penetrans. The PCA gave a loading plot for the soil variables as described earlier and a score plot for the samples gathered in the proportion classes previously defined. A Newman and Keuls test was performed on the soil data according to the projection of the proportion classes on the two PC1 and PC2 eigenvalues in order to confirm the statistical differences between them. The statistical analyses were performed with the ADE-4 multivariate analysis and graphical display software (Thioulouse et al., 1997).

3. Results 3.1. Comparisons between countries Soil characteristics. Comparing the mean soil characteristics analysed in each country (Table 3), soils from Ecuador had a higher proportion of clay particles than soils from Senegal that are characterised by sandy particles. Soils from Burkina Faso and Trinidad and Tobago have balanced textures. Those from Crete have a high proportion of coarse sand with less silt. Despite the soil N – P –K status is influenced by mineral and organic amendments, the soils from Ecuador were mineral rich compared with those from Senegal which were very poor. The EC, which depends on the clay nature and on the ionic adsorption, and the Wp4, which depends on the soil structure and on the clay architecture, are consistently higher in Ecuador than in Burkina Faso, Trinidad and Tobago, Crete and especially Senegal. All the pHs are similar and range between 6.3 and 7.3. Occurrence of Meloidogyne spp. and P. penetrans. The soil J2 populations of Meloidogyne spp. in the samples where P. penetrans occurred were much greater in Ecuador and Burkina Faso than in Crete, Trinidad and Tobago, and especially Senegal. There is a clear linear correlation between the proportion of the J2s infested by P. penetrans and the total population of J2s (Fig. 1). Statistical structure of the samples. PCA of the data sets for soil variables including physico-chemical and nematodes factors were performed. Loading (A) and score (B) plots are shown in Fig. 2. The fraction of variance accounted for by the first and the second PC are 43 and 10.35% (eigenvalues), respectively. As shown by the loading plot, the first axis PC1 relates to the texture: fine and coarse sands [FSa and CSa] at the positive end; fine silts [FSi] and clays [Cla], and consequently the wilting point [Wp4] and the EC at the negative. The second axis PC2 relates to organic matter [C and N] and nematode populations [Me] with its positive values, and to conductivity [Con] and phosphorus [P] with the negative values. The score plot of the samples shows that most of them

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Table 3 Mean physico-chemical characteristics of the soils in the different countries Characteristics Texture (%) Clays Fine silts Coarse silts Fine sands Coarse sands Minerals (meq%) Ca Mg Na K Cl SO4 HCO3 Al P (‰) Organic matter (‰) C N pH EC (meq%) Conductivity (mS) Wp4 (%) Meloidogyne spp. juveniles Total (dm23) Infested with P. penetrans (%)

Burkina Faso

Crete

Ecuador

Senegal

18.4 14.1 16.0 27.3 23.2

18.9 13.0 5.4 16.1 46.2

30.2 24.9 11.8 18.8 11.3

4.6 1.5 3.3 49.7 41.5

18.4 18.5 18.9 29.9 12.9

7.03 2.73 0.09 0.5 0.09 0.2 0.19 0.38 117.4

19.57 2.35 0.98 1.11 0.72 2.25 0.7 0.34 571.5

19.9 6.05 0.77 1.45 0.24 0.65 0.39 0.53 139.7

3.1 0.68 0.34 0.14 na na na na 186.1

6.96 1.28 0.15 0.65 0.15 0.41 0.27 0.49 360.5

11.2 1.1 6.4 8.3 0.33 7.7

18.1 1.8 7.3 13.1 0.8 6.4

20.8 2.1 6.7 25.3 0.39 16.6

5.0 0.7 6.3 3.4 0.22 2.2

14,719 24.4

6940 14

22,944 41.9

2456 4.7

Trinidad and Tobago

13.1 1.5 6.6 9.2 0.3 7.4 3484 15.08

na ¼ not analysed.

could be arranged along the PC1 axis, except those from Crete and Ecuador. This was especially so for Senegal where all the samples are located at the positive end of the first axis PC1. In contrast, more of

Fig. 1. Correspondence between the proportion of Meloidogyne spp. juveniles infested by P. penetrans and the total number of juveniles in soils from the different countries (S ¼ Senegal; C ¼ Crete, T ¼ Trinidad and Tobago; B ¼ Burkina Faso; E ¼ Ecuador; bars represent standard errors; — ¼ regression line between the data means of each country).

the Ecuador samples are located at the negative end. Those from Burkina Faso, Crete and Trinidad and Tobago are located in the middle. This demonstrates that the Senegal soils analysed are the sandiest, whereas the Ecuador soils have the most clay and the soils from Burkina Faso, Trinidad and Tobago and Crete are intermediate. On the second axis PC2, the samples from each country are evenly distributed both positively and negatively, except for those from Crete, which are all negative (due to very high phosphorus concentrations). As the samples from Burkina Faso, Trinidad and Tobago, and Senegal are in the middle of the second axis, organic matter and minerals do not define them. In contrast, the soils from Ecuador can be divided into two large classes: those, which are more associated with higher organic matter (most of them were sampled in the Highlands and the Galapagos) whereas those, which are more associated with higher mineral contents (most of them were sampled in the Coast and Orient regions). Correspondence between the soil texture and the proportion of nematodes infested by P. penetrans. The proportion of J2s infested by P. penetrans was correlated with the texture gradient defined by the axis PC1 (Fig. 3): the proportion of infested J2s was increased by the content of fine particles (clays and fine silts).

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Fig. 2. PCA loading plot for the soil factors (A) and score plot for the soil samples (B).

3.2. Correspondences between soil factors and nematode parasitism in each country Only samples from Burkina Faso, Ecuador and Senegal were kept in this study because only relatively few were available from Crete and Trinidad and Tobago. In Ecuador. The PCA analysis of the soil factors indicated (Fig. 4(A)) that most of the variability in levels of infestation by P. penetrans could be explained by a texture gradient (PC1 ¼ 34%) and secondly by an organic matter gradient (PC2 ¼ 14.1%). Nematode population density [Me] was not determinant. However, as most of the soil samples have high clay or silt and a high organic matter content, the distributions of the samples are centred on the origins of the two axes PC1 and PC2 (Fig. 4(B)). So, the proportion of juveniles infested by P. penetrans does not

depend on the soil factors analysed (no significant differences). In Burkina Faso. The PCA analysis of the soil factors showed (Fig. 5(A)) that most of the variability of the data could be explained by a texture –organic matter gradient (PC1 ¼ 35.6%) with magnesium [Mg] and calcium [Ca] positively linked to the clay content, and secondly by an ionic gradient (PC2 ¼ 12.4%). The corresponding distributions of the samples are all arranged along the PC1 axis. The proportion of infested J2s increases as the clay and organic matter content increase in the soils, without any difference between the a and b proportion classes which are concentric (Table 4). According to the axis PC2, the proportion classes can be arranged in two groups: those with a low proportion of infested J2s (a and b ) and those with a higher proportion (c and d ). The former is associated with the highest chloride [Cl] and sulphate [SO4] concentrations. In Senegal. The PCA analysis of the soil factors showed (Fig. 6(A)) that most of the variability of the data could be explained by a texture gradient (PC1 ¼ 39.5%) with magnesium [Mg] and EC certainly positively linked to the clay content, and secondly by a phosphate—organic matter gradient (PC2 ¼ 22.4%). The corresponding distributions of the samples according to the proportions of infested J2s are all arranged along the PC1 axis. The proportion of infested J2s increases as the fine particle content of the soil increase (Table 5). On the PC2 axis, J2s infested by P. penetrans are more frequent when the phosphate [P] and organic matter [C and N] contents are high.

4. Discussion

Fig. 3. Correspondence between the percentage of juveniles of Meloidogyne spp. infested by P. penetrans and the mean texture of the soils (% of particles) in the different countries (S ¼ Senegal; C ¼ Crete, T ¼ Trinidad and Tobago; B ¼ Burkina Faso; E ¼ Ecuador; bars represent standard errors; — ¼ log curve fits between the data means of each country).

M. incognita and M. javanica were the most widespread root-knot nematode species in all countries (Trudgill et al., 2000). Then, the infestation of the Meloidogyne spp. populations by P. penetrans cannot be ascribed to the nematode-bacterium specificity only (Oostendorp et al.,

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Fig. 4. PCA loading plot for the soil factors (A) and score plot for the samples according to their Meloidogyne spp. populations infested by P. penetrans (B): Ecuador data.

Fig. 5. PCA loading plot for the soil factors (A) and score plot for the samples according to their Meloidogyne spp. populations infested by P. penetrans (B): Burkina Faso data.

Fig. 6. PCA loading plot for the soil factors (A) and score plot for the samples according to their Meloidogyne spp. populations infested by P. penetrans (B): Senegal data.

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Table 4 Correspondence between the proportion of infested juveniles (1 , a # 10 , b # 20 , c # 50 , d ) and the significative soil characteristics on the PC1 and PC2 eigenvalues of the PCA: Burkina Faso data (data followed by the same letters are not significantly different, p (Newman and Keuls) , 0.05) Infested juveniles (%)

Soil characteristics

Proportion classes

Mean (SD)

Clays (%)

According to PC1 a þ b ¼ 1–20 c ¼ 21– 50 d . 50

10.7 (1.1) 34.2 (2.3) 79.4 (3.9)

12.2 c 15.4 b 22.7 a

According to PC2 a þ b ¼ 1–20 c þ d . 20

10.7 (1.1) 50.5 (4.9)

P (meq) 106.0 a 112.8 a

1990; Davies and Dansk, 1993). The apparent densitydependent relationship between Meloidogyne spp. and levels of infestation by P. penetrans is important, as it appears consistent in all the countries. But, despite this host – parasite dependence (Ciancio and Bourijate, 1995; Diop et al., 1996), this study suggests that the soil physicochemical characteristics can interact on proportions of nematode infestation by P. penetrans. The analysis among the countries points out the interaction of soil factors, especially of the exchange complex with the clay content and with its associated components (e.g. wilting point and exchange minerals). The implication of the clay content on the proportion of Meloidogyne spp. juveniles infested by P. penetrans may be strengthened by an increase in the organic matter content. Analyses made within each country give more information. In Burkina Faso and in Senegal, the proportion of the infested nematodes would be influenced by soil texture with infested J2s being more numerous in light soils than in heavy ones. But, the results obtained from Senegal point out that the presence of fine particles (clays or organic matter) seems to be essential in increasing the abundance of P. penetrans in sandy soils (Mateille et al., 1995). That is confirmed in Ecuador, where the texture of the soil samples analysed is more balanced between fine and coarse particles, and them would be more homogeneously fitted for nematode infestation by P. penetrans.

Fine þ coarse sands (%)

Organic matter (%)

57.6 a 56.3 a 40.7 b

1.3 c 1.7 b 2.2 a

Cl (meq) 0.11 a 0.08 b

SO4 (meq) 0.26 a 0.16 b

In fact, as it was shown in Senegal, despite a very low abundance of P. penetrans, the bacterium occurred in three different soils distinguished according to their texture (Mateille et al., 1995). The most unfitted soils are characterised by a lack of aggregates (e.g. coarse soils), or pure clays with no porosity (e.g. soils from the Senegal valley from which P. penetrans was rare despite the widespread occurrence of root-knot nematodes). In coarse soils subjected to heavy rainfall or irrigation, the spores of P. penetrans may be washed down the profile and lost (Dabire´ et al., 1996), as it occurs with many soil microorganisms (Gannon et al., 1991; Hornberg et al., 1992). In compacted soils, the migration of Meloidogyne J2s may be impaired (Prot, 1978), and so attachment of the spores of P. penetrans is reduced (Mateille et al., 1996). Concurrently, other factors in regard to the soil structure could be concerned. The EC and other factors affecting the soil solution could be involved in balance mechanisms between the adsorption of the spore of P. penetrans on the soil particles and their availability for attachment on nematodes (Mateille et al., 1996). In Ecuador for example, the potassium/EC association would result in more structured clays (illites—swollen clays) than those found in the other investigated countries, which are closer to kaolinites, helping a more homogeneous spatial distribution of the spores in the soil. So, despite that the infestation of the juveniles of

Table 5 Correspondence between the proportion of infested juveniles (1 # a , 5 # b , 10 # c ) and the significative soil characteristics on the PC1 and PC2 eigenvalues of the PCA: Senegal data (data followed by the same letters are not significantly different, p (Newman and Keuls) , 0.05) Infested juveniles (%)

Soil characteristics

Proportion classes

Mean (SD)

Clays (%)

According to PC1 A ¼ 1–4 b ¼ 5–9 c $ 10

1.6 (0.4) 6.1 (0.6) 42.2 (12.8)

3.7 b 3.9 b 5.1 a

According to PC2 a ¼ 1–4 bþc$5

1.6 (0.4) 24.1 (9.9)

P (meq) 124.2 b 208 a

Fine þ coarse silts (%)

Coarse sands (%)

2.2 b 5.4 a 5.4 a

48.6 a 34.2 b 40.8 b

Organic matter (‰) 3.9 b 5.4 a

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Meloidogyne spp. depends on the production of the spores of P. penetrans (obligatory parasitism), the influence of the soil environment on infestation mechanisms is clearly revealed. Especially with the unculturable bacteria P. penetrans, as inoculative or inondative biocontrol techniques are limited by its production, a rational management of the environmental factors would be able to improve its efficiency. Mainly supported by the management of crop systems and cultural practices (soil tillage, rotations, etc.), it would allow conceiving and developing integrated meso-biocontrol techniques compatible with sustainable agriculture.

Acknowledgments This study was supported by a grant from the EC Project STD 3 no. TS3 p CT92-0098: Biocontrol of damaging rootknot nematode (Meloidogyne spp.) pests of staple food and cash crops by including suppressive soils with the bacterial parasite P. penetrans.

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