EFFECTS OF DIFFERENT ORGANIC AMENDMENTS ON BANANA PARASITIC AND SOIL NEMATODE COMMUNITIES

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Soil, plant material, and organic amendments

Soil was collected in the 0–20 cm layer of a banana field of Guadeloupe (16°N, 61°W) in June 2008. This soil was classified as Haplic Nitisol (IUSS Working Group WRB, 2006), comprising 80% clay; 14% silt; 6% sand; pH (H2O) 5.45; 1.80% organic C and 0.16% total N. After sieving at 13mm, the soil was hand-blended with organic amendments, namely sugarcane bagasse, sewage sludge, sugarcane refinery sludge and plant residues, before being placed in 3L-pots (about 3kg dry soil per pot). Bagasse is a by-product coming from the sugarcane pressed to produce rum. Sugarcane refinery sludge (also called filter mud) is a major cane processing waste, recovered from press and vacuum filters when sludge from the clarification process by Calcium salts is dewatered. Plant residues mainly came from the plant pruning in Guadeloupe and had been crushed in about 5-cm piece before incorporation. The biochemical characteristics of these amendments are shown in Table 1. Into each treatment, we placed fresh amount of amendment so that 60g dry matter was exactly blended to the soil in each 3L-pot. The pots were then placed in a greenhouse for an incubation period of three weeks. At the end of this incubation period, a banana seedling issued from tissue culture (Musa acuminata, subgroup Cavendish, cv. Grande-Naine) was planted in each pot. Bananas were then grown for 10 weeks. Soil moisture was maintained near and always below field capacity in order to avoid any water stress, preventing any soil anoxia and drainage: three times a week, each pot was weighed, and distilled water was supplied to maintain soil water content at field capacity during the whole experiment, and every three weeks, the pot reference weight was corrected by measuring the fresh biomass of one plant per treatment.

Experimental design

The experiment compared the effect of five treatments: sugarcane bagasse (SCB), sewage sludge (SES), sugarcane factory sludge (SCS), also called filter mud, plant residues (PLR), and a control without amendment (CTL). Each treatment was replicated in 12 pots, randomly located in the greenhouse. Three pots were used to assess soil and banana plant mineral N status. Nine others were used to assess banana plant growth, banana root necrosis, and plant-parasitic nematode abundances in banana roots. Measurements were made at four dates: (1) stage O (original stage), (2) three days after amendment application, stage A, (3) three weeks later, stage P (planting of banana), and (4) stage F (final stage), ten weeks later.

Soil mineral and microbial nitrogen

Soil microbial biomass and mineral N were measured for each treatment from 3 soil samples taken from 3 pots randomly chosen. Soil microbial biomass, expressed as microbial N, was measured according to the fumigation-extraction method (Amato and Ladd, 1988). Soil mineral N was also measured by colorimetry after N extraction with a KCl 1M solution (AFNOR, 2007).

Plant growth and banana N content

At stage P, banana dry matter and plant N content were determined on 6 plants. At stage F, for one given treatment, shoot dry matter was determined after oven drying at 60°C for 4 days, on the twelve banana plants. Root dry matter, measured after oven drying at 60°C for 4 days, was determined on three banana plants randomly chosen, from which we determined N plant content (shoot plus root parts). Root fresh matter was measured on the nine other bananas.

Banana available N

Available N for plant (Navailable) was determined according to the following equation:
Navailable = Nplant uptake + NminF with, = Plant N content at stage F – plant N content at stage P, NminF = Pot (i.e. soil and amendment) mineral N at stage F.

Plant-parasitic nematodes and root necrosis analysis

At stage F, each root system was scanned, with a resolution of 600 dpi. WinRHIZO 2009a Software (Regent Instruments Canada Inc.) was used to calculate the surface of healthy and necrotic part of the root system after defining the color classes characterizing each part. The color of root necrosis due to soil-borne pathogens turned from reddish to black while healthy root were white. The necrosis rate was expressed as necrotic surface area over total root surface area.
Then, the nematodes in the roots were extracted using a centrifugal-flotation technique (modified from Coolen and D’Herde, 1972). Root systems were crushed in a blender. This suspension was poured on a sieve column and then abundantly washed. The later residues through the last sieve (32µm) were gently washed in a 500ml centrifuge tube. A first centrifugation (1500g for 5min) discarded the supernatant. The centrifuge tube was then filled with a solution of MgSO4 (1.17g/L). A second centrifugation (1500g for 5min) was performed and the supernatant containing nematodes was collected on a 5µm sieve. Plant-parasitic nematodes were counted and identified three times in 1ml aliquots out of a 100-ml volume using a binocular microscope (X400). For a given species, we calculated two variables: nematode abundance, as the total number of nematodes in the whole root system and nematode root density, as the number of individuals per 100g fresh root. Nematode abundance was used to assess the effect of treatments on nematode community regulation in the microcosm. We did not use nematode density in the roots although this measure is often used to assess plant-parasitic nematode in the roots, especially for field experiments. In fact, using root densities introduces a second variable (i.e. root biomass) that might vary between the treatments: root density was not thus a stable indicator of nematode regulation. Nematode density was used to explain root damages assessed through necrotic rate.

Statistical analysis

Soil N data and nematode data were log(x+1)-transformed to normalize variances prior to analysis. ANOVA, Tukey’s multiple comparison (P value threshold: 0. 05), and regression were performed using XL STAT (version 2009 6 02, Addinsoft). Lastly, total plant-parasitic nematode abundance was submitted to an analysis of covariance (ANCOVA) in which organic amendment treatment was the qualitative factor and fresh root biomass was the quantitative co-factor, performed with XLSTAT (Version 2009.6.02, Addinsoft).

Results

Changes in soil microbial N and mineral N

Changes on soil microbial N differed between treatments (Fig. 1). At stage A, the highest soil microbial biomass N was observed for treatments with sewage sludge (SES) and sugarcane refinery sludge (SCS) compared to treatments with bagasse (SCB) and plant residues (PLR) and the control (CTL). At stage F, soil microbial biomass N in SES and SCS stayed higher than that of the control even though SES treatment values fell greatly. In SCB and PLR, soil microbial biomass N overcame that of the control.
As shown in Fig. 2, treatment SES had the highest mineral N content at stage A and was significantly higher than the initial value (stage O). Conversely, mineral N contents were significantly lower in SCB, PLR, and SCS and were significantly lower than the initial value. At stage F, mineral N content did not differ between PLR, SCB and CTL, and mineral N content in SCS overcame that of the control. Mineral N content in SES diminished greatly but remained the highest.

Banana growth and plant-parasitic nematodes

Banana plant grew the best in treatments SES and SCS (respectively 20.2g and 14.9g), in contrast, plants in SCB exhibited the smallest biomasses (2.2g) (P<0.0001). Their growth in SCB was restrained in comparison with the control banana plants (P<0.0001). Banana plant growth was closely linked to available mineral N (R2=0.98 by a sigmoidal adjustment, with the formula y=20.1/ (1+e (2.95-0.002x)). Banana growth in SES attained the plateau of the regression (20.1g) suggesting a maximized plant growth in this treatment.
Relationship between plant-parasitic nematode abundance within whole root system and root biomass is presented in Fig 3. This figure shows that for higher root biomasses than those of the control, SES exhibited plant-parasitic nematode abundances near to those of the control. Treatment SCS exhibited lower plant-parasitic nematode abundance within higher root biomass than the control, whereas SCB and PLR exhibited lower nematode abundance within lower root biomass than those of the control.
The effects of organic material treatment and of root biomass on the abundances of plant-parasitic nematodes were evaluated by an ANCOVA without interaction (P value of variance analysis <0.0001). We first built a model of ANCOVA with interaction between the two factors in which the effects of interaction were not significant (data not shown). According to the ANCOVA without interaction, the effect of the root biomass on the abundances of plant-parasitic nematodes was not significant (P value = 0.14); in contrast, the effect of organic material treatment was highly significant (P value<0.0001).
The abundances within the root system and the root densities of plant-parasitic nematodes are presented in Table 2. In treatments SCB, PLR and SCS, their total abundances were significantly lower than in the control and the treatment SES. We also observed differences between species: while Pratylenchus coffeae was significantly suppressed in SCB, SCS, and PLR, the abundance of Meloidogyne spp. was lowered only in SCB. The root densities of plant-parasitic nematodes were also lower in treatments SCB, SCS and PLR than in SES and the control; even if the root density of P. coffeae in SES were lower than the control.

Root necrosis

Relationship between root necrosis rate and density of plant parasitic nematodes in the roots is presented in Fig. 4. This figure shows that for a given density (lower than 10,000 individuals for 100 g DM roots), the necrosis rate varied with the treatment (SCB, SCS, and PLR). For higher densities (SES and the control) the necrosis rates were high.

Discussion

Nitrogen dynamics and banana plant growth

Our results showed that the changes in banana growth that were observed between the treatments were highly related to the quantity of available N added. This relationship can be adjusted with a sigmoidal function which fits well conceptually with plant N limitation at low available N quantities. The greatest N supply and microbial biomass were obtained with sewage sludge (SES). Adding sewage sludge to the soil provided high quantities of mineral N, already present in this organic material at its application. Moreover, this amendment was probably full of microorganisms, whose activity caused considerable net N mineralization and whose mortality released N during the trial. These results are supported by those of Stamatiadis et al. (1999), who observed great stimulation of microbial activity and increased net N mineralization after sewage sludge addition. The fast disappearance of microbial biomass with the SES treatment was probably due to fast depletion of C resources from a low C: N ratio-substrate. In contrast, treatment with sugarcane bagasse (SCB) supplied almost no N to banana plants. Sugarcane bagasse also exhibited a high C: N ratio. The microbial biomass that developed throughout the trial with this treatment took soil mineral N, causing N immobilization under organic N (« N deficiency » phenomenon) that limited banana growth. Between these two extremes, the available N quantity at the end of the trial with plant residues (PLR) was equivalent to that of the control. This amendment exhibited a C: N ratio between that of SES and SCB and a high and recalcitrant-degradation lignin-like fraction (Thuriès et al., 2002). These characteristics explain the low N release after an immobilization period. Lastly, sugarcane sludge (SCS) also led to N immobilization, which then permitted higher N release than that of PLR, because of a more favorable biochemical composition.

Nematode regulation and parasitism impacts on banana plants

Our results showed that only three amendments induced nematode regulation, namely sugarcane bagasse, sugarcane refinery sludge and plant residues. In the conditions of our experiment, the root system dimension was not a major factor explaining the plant-parasitic nematode abundances in the roots, although others authors showed a relationship between populations of plant-parasitic nematodes and plant development stage (e.g. Villenave et al., 2010; Yeates, 1987). As a result, even the smallest root system might be amply sufficient to sustain the growth of plant-parasitic nematode populations. The observed differences in nematode abundance between the treatments were due to organic material inputs.
Our results showed also that each amendment had its own impact on the populations of plant-parasitic nematodes. The three regulated taxa, to wit P. coffeae, R. reniformis and Meloidogyne spp., did not respond the same way with the three regulatory organic materials: sugarcane bagasse exhibited a generalized effect whereas sugarcane sludge was the most selective treatment. Pattison et al. (2006), like us, showed a variability of effects after different C compound inputs (such as lignins and cellulose) on Meloidogyne spp. and R. similis. Our results on regulation efficiency of different amendments are supported by Pattison et al. (2006), who found no regulation of banana parasitic nematodes with sewage sludge and by Stirling et al. (2005) who also showed nematode regulation after sugarcane trash inputs. In our experiment, SCS appears to be the most efficiency amendment, decreasing parasitism pressure and enhancing plant growth. The mechanisms of plant-parasitic nematode regulation were not presented in this paper, except that of plant resources. Many different regulation mechanisms can occur, which can be biologic, such as predation by nematodes and fungi, or induced resistance of the plant, or abiotic, such as the release of ammonia (see the review by Oka, 2010). Our results also highlighted the importance to take account the biochemical composition of organic amendments when regulation mechanisms are studied.

Table of contents :

INTRODUCTION
CHAPITRE 1: EFFETS DE QUATRE MATIÈRES ORGANIQUES BRUTES SUR LE CONTRÔLE DES NÉMATODES PARASITES DU BANANIER ET SUR LES COMMUNAUTÉS DE NÉMATODES DANS LE SOL : ESSAI EN MICROCOSME
PARTIE 1: EFFECTS OF ORGANIC AMENDMENTS ON PLANT-PARASITIC NEMATODE POPULATIONS, ROOT DAMAGE AND BANANA PLANT GROWTH
1.1.1. Abstract
1.1.2. Introduction
1.1.3. Materials and methods
1.1.3.1. Soil, plant material, and organic amendments
1.1.3.2.Experimental design
1.1.3.3. Soil mineral and microbial nitrogen
1.1.3.4. Plant growth and banana N content
1.1.3.5. Banana available N
1.1.3.6. Plant-parasitic nematodes and root necrosis analysis
1.1.3.7. Statistical analysis
1.1.4. Results
1.1.4.1. Changes in soil microbial N and mineral N
1.1.4.2. Banana growth and plant-parasitic nematodes
1.1.4.3. Root necrosis
1.1.5. Discussion
1.1.5.1. Nitrogen dynamics and banana plant growth
1.1.5.2. Nematode regulation and parasitism impacts on banana plants
PARTIE 2: EFFECTS OF DIFFERENT ORGANIC AMENDMENTS ON BANANA PARASITIC AND SOIL NEMATODE COMMUNITIES
1.2.1. Abstract
1.2.2. Introduction
1.2.3. Materials and methods
1.2.3.1. Soil, plant material, and organic amendments
1.2.3.2. Experimental design
1.2.3.3. Chemical and biochemical characterization of the amendments and N-NH4 + in soil
1.2.3.4. Nematodes in roots
1.2.3.5. Nematodes in soil
1.2.3.6. Nematode community index
1.2.3.7. Statistical analysis
1.2.4. Results
1.2.4.1. Main chemical and organic components of the four amendments
1.2.4.2. Plant-parasitic nematodes in banana roots
1.2.4.3. Soil nematode community composition
1.2.4.4. Nematofauna indices
1.2.5. Discussion
1.2.5.1. Suppression of plant-parasitic nematodes in roots and characteristics of the organic amendments
1.2.5.2. Impacts of amendments on soil nematode community structure
1.2.5.3. Mechanisms of plant-parasitic nematode suppression
1.2.6. Conclusion
CHAPITRE 2: EFFECTS OF COMPOSTS ON BANANA GROWTH, PLANT-PARASITIC NEMATODE POPULATIONS, AND SOIL NEMATODE FOOD WEB
2.1. Abstract
2.2. Introduction
2.3. Materials and methods
2.3.1. Trial location, experimental design and pot management
2.3.2. Chemical and biochemical characterization of the amendments
2.3.3. Soil sampling
2.3.4. Plant growth and available mineral N in the pots
2.3.5. Soil microbial biomass
2.3.6. Nematode extraction and identification in the roots
2.3.7. Nematode extraction and identification in soil
2.3.8. Statistical analysis
2.4. Results
2.4.1. Chemical and biochemical characteristics of amendments
2.4.2. Changes in plant growth with treatments
2.4.3. Plant growth and plant-parasitic nematodes in the roots
2.4.4. Microbial biomass and nematode community structure in soil
2.5. Discussion
2.5.1. Effects of composts on plant-parasitic nematode control
2.5.1.1. Effects of composts on the quantity of root resources
2.5.1.2. Compost effects on the size of populations of banana parasitic nematodes
2.5.1.3. Compost effects on specific composition of plant-parasitic nematodes in the roots
2.5.2. Compost effects on soil nematode communities
2.5.2.1. Direct effects of organic material application on nematode community structure
2.5.2.2. Changes in nematode food web structure
CHAPITRE 3 : VARIABILITÉ SPATIALE ET EFFET DE LA STRUCTURE DU SOL SUR LES POPULATIONS DE NÉMATODES PARASITES DU BANANIER ET LES COMMUNAUTÉS DE NÉMATODES DANS LE SOL À L’ÉCHELLE DU PROFIL CULTURAL
3.1. Introduction
3.2. Matériel et méthodes
3.2.1. Site expérimental et mise en place de l’essai
3.2.2. Description des profils culturaux et échantillonnage
3.2.3. Densité, nécrose et diamètre des racines
3.2.4. Nématodes dans les racines et dans le sol
3.2.5. Variables descriptives de la structure du sol et de la matière organique
3.2.6. Analyses statistiques
3.2.6.1. Statistiques inférentielles
3.2.6.2.Analyses en composantes principales
3.2.6.3. Analyses de co-inertie
3.3. Résultats
3.3.1. Caractéristiques structurales et spatiales des profils
3.3.1.1. Description morphologique des profils
3.3.1.2. Analyse des profils
3.3.1.3. Densité et diamètre des racines
3.3.2. Nématodes phytoparasites et nécroses
3.3.2.1. Variabilité spatiale du taux de nécrose et de la densité des nématodes phytoparasites dans les racines
3.3.2.2. Structure des populations de nématodes phytoparasites dans les racines
3.3.3. Variabilité spatiale des communautés de nématodes dans le sol
3.3.3.1. Structure taxonomique des nématodes phytoparasites dans le sol
3.3.3.2. Structure taxonomique des nématodes « libres » dans le sol
3.3.4. Variabilité des indices nématologiques
3.3.5. Relation sol-nématodes
3.3.5.1. Analyse de co-inertie entre les nématodes phytoparasites dans les racines et les variables du milieu
3.3.5.2. Analyse de co-inertie entre les nématodes phytoparasites dans le sol et les variables du milieu
3.3.5.3. Analyse de co-inertie des taxons de nématodes « libres » du sol et des variables du milieu
3.4. Discussion
3.4.1. Effets de la variabilité de la structure du sol et de l’apport de compost sur la composition et la structure des communautés de nématodes
3.4.1.1. Effet de la structure du sol
3.4.1.2. Effet de l’apport de compost
3.4.2. Effets de la variabilité spatiale des ressources sur la composition et la structure des communautés de nématodes
3.4.2.1. Effets sur les nématodes phytoparasites dans les racines et dans le sol
3.4.2.2. Effets sur les nématodes « libres » dans le sol et sur le fonctionnement du réseau trophique des nématodes
CONCLUSION GÉNÉRALE
4.1. Principaux résultats acquis au cours de la thèse et limites de ce travail
4.1.1. Effet des apports de matières organiques brutes et compostées sur le contrôle biologique des populations de nématodes
4.1.2. Mécanismes mis en jeu dans les contrôles observés.
4.1.3. Paramètres du milieu qui structurent la répartition des nématodes « libres » et parasites dans le sol, à l’échelle du profil cultural
4.2. Perspectives de recherche
RÉFÉRENCES BIBLIOGRAPHIQUES

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