Diversity of plant parasitic nematodes characterized from fields of the French national monitoring programme for the Columbia root-knot nematode.

Plant parasitic nematodes are highly abundant in all agrosystems and some species can have a major impact on crop yields. To avoid the use of chemical agents and to find alternative methods to manage these pests, research studies have mainly focused on plant resistance genes and biocontrol methods involving host plants or natural enemies. A specific alternative method may consist in supporting non-damaging indigenous species that could compete with damaging introduced species to decrease and keep their abundance at low level. For this purpose, knowledge about the biodiversity, structure and functioning of these indigenous communities is needed in order to carry out better risk assessments and to develop possible future management strategies. Here, we investigated 35 root crop fields in eight regions over two consecutive years. The aims were to describe plant parasitic nematode diversity and to assess the potential effects of cultivation practices and environmental variables on communities. Community biodiversity included 10 taxa of plant parasitic nematodes. Despite no significant abundance variations between the two sampling years, structures of communities varied among the different regions. Metadata collected for the past six years, characterizing the cultural practices and soils properties, made it possible to evaluate the impact of these variables both on the whole community and on each taxon separately. Our results suggest that, at a large scale, many variables drive the structuration of the communities. Soil variables, but also rainfall, explain the population density variations among the geographical areas. The effect of the variables differed among the taxa, but fields with few herbicide applications and being pH neutral with low heavy metal and nitrogen concentrations had the highest plant parasitic nematode densities. We discuss how these variables can affect nematode communities either directly or indirectly. These types of studies can help to better understand the variables driving the nematode communities structuration in order to support the abundance of indigenous non-damaging communities that could compete with the invasive species.

Introduction

Plant parasitic nematodes (PPNs) are soil organisms that feed on plant tissues. A limited number of these nematodes can induce economic losses by decreasing yield or causing visual damage, making the products unsuitable for sale [1]. Even though most PPNs are not damaging to crops at the abundances commonly found in agricultural soils, some taxa are known to have a major negative impact. Jones et al. [2] listed the 10 most important plant parasitic nematodes, among which Meloidogyne spp. (also called root-knot nematodes) are in first place. In particular, the Columbia root-knot nematode, Meloidogyne chitwoodi, represents a threat to temperate agriculture, mainly for root crops [2]. In order to manage this quarantine pest in fields where it has been detected, several studies have focused on non-host and resistant plants [3,4], or on biocontrol strategies [5]. However, studies focusing on preventive strategies, such as preventing the establishment of the damaging nematodes in fields where they are not yet present, are lacking.

The biodiversity of indigenous PPN communities can play a major role in the establishment success of a newly introduced species, since indigenous species could be potential antagonists to the introduced one. For instance, competition for roots between PPN species among communities, has been observed [6-9]. In particular, Garcia et al. [9] showed, in a greenhouse experiment, that the risk of establishment of M. chitwoodi increases in less diverse and less abundant PPN communities. As a result, knowledge about the biodiversity and factors affecting indigenous PPN communities in root crop fields is needed. By extension, such knowledge could provide an ecological risk assessment tool that could be useful to improve the choice of the fields to sample in the framework of the French programme for monitoring the Columbia root-knot nematode not only considering the susceptible host plant but also on the indigenous PPN communities.

In agro-ecosystems, that are shaped by frequent anthropogenic disruptions, the structure and functioning of PPN communities depend mainly on crop management practices [10-13], but also on environmental conditions [12-16]. Intensive profound tillage is probably the most commonly studied cultivation practice and it has been shown that this tillage practice usually has a negative impact on PPN populations [10,17]. Cultivation practices are known to change the physico-chemical properties of the soil [18,19], which also affects the nematodes in the soil, by modifying the metabolism, movement or food accessibility for example [13,14,16]. However, studies often focus on a few environmental conditions or crop practices at the same time (e. g.: profound tillage, soil texture, or heavy metal content) [11,14,15]. There are, to our knowledge, only a few studies dealing with a larger number of variables and/or at broad spatial scales [20,21]. In the present study, we sampled fields that are part of the French monitoring programme for M. chitwoodi in several geographical areas in order to (i) describe the diversity and abundance of PPN communities in fields including a root crop in the crop rotation, (ii) evaluate whether cultivation practices identified in the literature at smaller scales in fact shape PPN communities at larger scale and (iii) assess the potential impact of environmental variables on PPN communities, including climate and physico-chemical conditions of soil.

Results

Characterization of the environment and PPN biodiversity

The sum of monthly precipitation and mean monthly temperatures were collected for the 12 months before each sampling date. Comparison tests showed that the climate was not significantly different over the two sampling years (W = 4.86, p-value = 0.283 and W = 638, p-value = 0.430 for precipitation and temperature comparisons, respectively).

For the 35 fields sampled in 2015, 10 taxa were identified and counted (9 genera and one family for which we were unable to identify nematodes to the genus level) (Table 1). PPNs identified were Helicotylenchus, Pratylenchus, Amplimerlinius, Paratylenchus, Criconemoides, Meloidogyne, Ditylenchus, Trichodorus, Heterodera and the Telotylenchidae family. Among these taxa, the most frequently found were Pratylenchus and Telotylenchidae, both identified in 91% of the fields (Table 1). Ditylenchus was found only in 3 fields (Table 1).

Table 1

Abundance comparison of the PPN communities found in 2015 and 2016 samplings.

	Mean ± Standard error in 100g of soil		Comparison 2015–2016			Prevalence (%)
	2015 (N = 35)	2016 (N = 31)	W	p-value	significance	2015	2016
Helicotylenchus	26.2 ± 9.2	67.9 ± 23.0	498.0	0.537		46	45
Pratylenchus	60.5 ± 10.4	67.3 ± 14.6	567.0	0.757		91	77
Amplimerlinius	14.5 ± 5.8	16.5 ± 5.5	536.5	0.935		37	35
Other Telotylenchidae	38.7 ± 6.7	84.9 ± 29.0	380.5	0.038	*	91	97
Paratylenchus	20.9 ± 6.7	38.3 ± 13.1	481.5	0.414		54	55
Criconemoïdes	2.6 ± 1.2	5.0 ± 2.6	524.0	0.746		20	23
Meloidogyne	13.4 ± 5.9	8.0 ± 3.0	608.0	0.353		49	35
Ditylenchus	0.5 ± 0.4	-	-	-		9	0
Trichodorus	1.3 ± 1.3	18.6 ± 6.0	325.0	2.09e-4	***	6	45
Heterodera (juveniles & eggs)	1312.6 ± 637.5	1685.6 ± 645.2	502.0	0.593		54	61
Shannon-Weaver Index	0.356 ± 0.037	0.413 ± 0.036	733.5	0.231

That data depict the mean of 35 and 31 fields surveyed in 2015 and 2016, respectively. “W” indicate the value of the Wilcoxon test;

* < 0.05;

** < 0.005;

*** < 0.001.

Prevalence indicate the percentage of fields where the taxon was found among all the fields of the same year.

Only nine taxa from among those mentioned above were identified in the 2016 samples from the 31 fields surveyed (Table 1). No additional taxon was found in 2016 compared to 2015, but Ditylenchus was no longer found in 2016. Pratylenchus and Telotylenchidae were still the most frequently observed PPNs. A comparison between the 2015 and 2016 communities showed a significant difference in Trichodorus abundance, increasing from 1.3 ± 1.3 individuals per 100 g of fresh soil in 2015 to 18.6 ± 6.0 in 2016 (W = 325.0, p-value = 2.09e-4) (Table 1). A significant difference was also observed between abundances of Telotylenchidae (increasing from 38.7 ± 6.7 individuals per 100 g of fresh soil to 84.9 ± 29.0, W = 380.5, p-value = 0.038). However, an overall PPN community comparison using the Shannon-Weaver diversity index, showed no significant differences between the 2015 and 2016 PPN communities (W = 733.5, p-value = 0.231).

Fields were sampled under the scope of the French monitoring program during the two consecutive years, no quarantine species was found in any of the samples and Meloidogyne sp. mentioned in this article correspond to non-quarantine species of Meloidogyne.

Effects of the variables on the whole PPN communities

A total of 6 transformation-based redundancy analyses (tb-RDAs) were performed, implemented for each analysis the cultivation practices of the previous years, moving backwards (i.e. sequential accumulation of the cultural practices over the five years prior to the samplings). These analyses aim to assess the effects of cultural practices and environmental variables on the overall community and a potential cumulative effect of the cultivation practices over the 5 past years (Figs 1 and S1). All variables were implemented to run each analysis without a preliminary selection step, since tb-RDA enables selection of only the significant variables with a permutation test. To ease the readability of the results, the first tb-RDA is presented here (Fig 1) (for which environmental variable and the cultural practices of the sampling year have been implemented) and the others tb-RDA (for which environmental variable and the cultural practices of the past years have been implemented) are available in supplementary data (S1 Fig).

Fig 1

Outputs of the tb-RDA for which the environmental variables and the cultivation practices of the sampling year have been implemented.

Arrows and bold text indicate the significant variables after a permutation test. Modalities of soils depend on the proportion of sand, silt and clay in it (SoilSaSiC = majority of sand, then silt then clay; SoilSiCSa = majority of silt, than clay, then sand; SoilSiSaC = majority of silt, then sand, then clay).

The first two axes represented on Fig 1 explain 31% of the variance of the dataset. Environmental variables (mainly soil properties) appear to be the main drivers of PPN communities. Rainfall, C/N ratio, soil texture and heavy metal concentration (Zn or Cu) are retained on the analysis. The arrow length (indicating the importance of the effect of the variable) of the C/N ratio or rainfalls in particular indicates a key role in structuring of the communities (Fig 1). For example, Helicotylenchus abundance present higher abundance in communities found in soil with a higher C/N ratio. No agricultural practices appeared on this analysis (Fig 1) but a cumulative effect of non-herbicide application and profound tillage of the past years is observed on the following tb-RDA (S1 Fig).

The field surface area also appears on the analysis and we chose to use the surface as a random effect in the following analyses to take into account that the sampling protocol were not fully adapted to the field surface area.

Many PPN taxa are quite close to the center of the factorial map (Fig 1), indicating that the retained variables, despite a significant impact on overall community structure, have less importance for each taxon individually. As a consequence, we performed generalized linear mixed models (GLMMs) for each PPN taxon, in order to assess the impact of the same variables on the abundance variation between fields.

Results of variable selection

Similarly to the tb-RDA, 6 multiple correspondence analyses (MCAs) were performed, adding the cultivation practices of the past years for each. The first two axes of each factorial map explained around 26% of the dataset variance. For the GLMM, we chose to retain only the variables showing at least two modalities on a minimum of two factorial maps. Hence, soil pH, Zn concentration in the soil, C/N ratio, and number of applications of herbicide products (present on 6, 5, 5 and 2 factorial maps, respectively were retained (factorial maps are available in supplementary data (S2 Fig)).

Effect of the selected variables

After the variables selection step, we used a model-averaging approach based on GLMM to test the effective impact of the selected variables on the abundance variation of the PPN taxa. Similarly to the previous analyses, we ran several GLMMs to test a potential cumulative effect of the past applications of herbicide products on the PPN communities. We implemented the four selected variables in the GLMMs. Sixteen first-order models were ranked for each taxon and year or year period. The sum of weight (SW) and 95% confidence interval (CI) were calculated for the variables present in the subset of models with a ΔAICc < 6. A summary of the results is presented in Table 2, while raw detailed results are available in supplementary material (S1 Table).

Table 2

Equations summarizing the model selection approach of the Poisson GLMMs for each taxa and each year periods.

Only significant variables are presented.

Taxon	Year period	Significative variables	Taxon	Year period	Significative variables
Helicotylenchus	n	Helico ~ C/N - Herbi	Criconemoides	n	Crico ~ C/N - Herbi
	n-1	Helico ~ - Herbi		n-1	Crico ~ - Herbi
	n-2	Helico ~ C/N		n-2	Crico ~ C/N
	n-3	Helico ~ - Herbi		n-3	Crico ~ C/N
	n-4	Helico ~ - Herbi		n-4	Crico ~ C/N
	n-5	Helico ~ C/N		n-5	Crico ~ C/N
Pratylenchus	n	Prat ~ - C/N - Zn	Meloidogyne	n	Melo ~ C/N + pH - Zn
	n-1	Prat ~ - C/N - Zn - Herbi		n-1	Melo ~ C/N + pH - Zn
	n-2	Prat ~ - C/N - Zn - Herbi		n-2	Melo ~ C/N + pH - Zn
	n-3	Prat ~ - C/N - Zn - Herbi		n-3	Melo ~ C/N + pH - Zn
	n-4	Prat ~ - C/N - Zn - Herbi		n-4	Melo ~ C/N + pH - Zn
	n-5	Prat ~ - C/N - Herbi		n-5	Melo ~ C/N + pH - Zn
Telotylenchidae	n	Telotyl ~ pH - Zn - Herbi	Trichodorus	n	Tricho ~ - Zn - Herbi
	n-1	Telotyl ~ pH - Zn + Herbi		n-1	Tricho ~ - Zn
	n-2	Telotyl ~ pH - Zn + Herbi		n-2	Tricho ~ - Zn - Herbi
	n-3	Telotyl ~ pH - Zn + Herbi		n-3	Tricho ~ - Zn
	n-4	Telotyl ~ pH - Zn + Herbi		n-4	Tricho ~ - Zn - Herbi
	n-5	Telotyl ~ - Herbi		n-5	Tricho ~ - Zn
Paratylenchus	n	Parat ~ C/N + pH	Heterodera	n	Hetero ~ Zn - Herbi
	n-1	Parat ~ C/N + pH		n-1	Hetero ~ Zn
	n-2	Parat ~ C/N + pH		n-2	Hetero ~ Zn - Herbi
	n-3	Parat ~ C/N + pH		n-3	Hetero ~ Zn - Herbi
	n-4	Parat ~ C/N + pH		n-4	Hetero ~ - Herbi
	n-5	Parat ~ - Herbi		n-5	Hetero ~ Herbi

“C/N” correspond to carbon/nitrogen ratio; “Herbi” correspond the number of applications of herbicide products over the time period considered; “Zn” correspond to the quantity of zinc in the soil; “Year period” indicate which year of cultural practices have been considered (i.e. “n” means the cultural practices of the sampling year, “n-1” means the cultural practices of the sampling year and the first past year etc.) to observe potential cumulative effect. No variables had a significant impact on Amplimerlinus population variations, explaining its absence in this table.

None of the considered variables impacted Amplimerlinius abundance in this study. One to three out of the four retained variables have appeared to significantly impact PPN abundance, depending on the taxa and the time period considered (Table 1). Soil pH–varying between 5.72 and 7.89 in our dataset–appears to positively impact the abundance of Meloidogyne, Paratylenchus and the Telotylenchidae while Zn concentration in the soil–ranging from 1.23 to 11.12 mg/kg–appears to negatively affect the abundance of Trichodorus, Meloidogyne, Telotylenchidae and Pratylenchus, for which the population abundance seemed to decrease in higher Zn concentration soils. However, Zn concentration appears to positively impact the abundance of Heterodera. Similarly, C/N ratio–ranging from 7.28 to 25.87 –appears to have a positive impact on Meloidogyne, Criconemoides, Paratylenchus and Helicotylenchus, but decrease Pratylenchus abundance. The application of herbicides, the only cultivation practice implemented in the GLMM following the MCA selection step, had a negative effect in the short or long term, depending on the taxon considered. PPN populations’ abundance were lower in fields where herbicide products were often used. The results are unclear concerning the Telotylenchidae as herbicides had a negative impact if the sampling year (short-term) or the 5 years before the samplings (longest-term) were considered. However, herbicides had a positive impact on Telotylenchidae if cultivation practices of the years before the samplings, and 2 to 4 years before the sampling were considered.

Discussion

PPN community biodiversity

This study describes the PPN communities in agrosystems including root crops in France. Sampling in eight geographical areas highlighted differences between communities due to environmental and cultural practices heterogeneity between the fields. The overall biodiversity, with 10 PPN taxa identified across the fields, is similar to other cereals and vegetables agrosystems studied in the literature [21-23]. The fields were chosen among those monitored as part of the French monitoring programme for the Columbia root-knot nematode, based on 2014 or 2015 crops (root vegetable crops), without information about crop rotation. It appears on the basis of crop management information collected from farmers that root crops were not very frequent in crop rotations, and farmers mainly grew wheat or maize, regardless of the region. Seven out of the ten taxa were present in at least 5 out of the 8 regions. The presence of the same PPN taxa in several regions could be due to their generalist feeding habits at the genus level and their plasticity regarding environmental conditions. Identified PPN taxa are mainly colonisers and are not particularly sensitive to environmental stress, explaining their presence in the majority of the regions [24,25]. However, one of the limitations here is that despite the large geographic scale investigated, it was not possible to sample numerous fields in each region. It is therefore not possible to statistically compare the communities between regions more deeply. Interestingly, despite homogeneity between 2015 and 2016 PPN communities, Trichodorus was the only genus substantially more abundant in 2016. This nematode feeds ectoparasitically mainly on sugar beet and carrot, and can induce economic issues in sandy soil due to reduced plant growth known as “docking disorder” [1,26]. Beet or carrot was grown in 2015 in the fields with high 2016 abundance of Trichodorus, whereas crops cultivated before 2015 were generally cereals. The presence of a suitable host plant could explain this increase in abundance. Furthermore, Trichodorus has a very large host range [27], including many weeds that could have been present in sampled fields despite the use of herbicide chemicals. Similarly, the significant increase in Telotylenchidae between the two sampling years could be due to the presence of a better host plant in 2016, whether the crop grown that year or weeds. The other PPN taxa were not significantly different between the two years, probably because of short crop rotations dominated by cereals.

The effects of variables on the overall PPN communities

In this study, we have observed mainly the impact of soil variables on the abundance variation of the PPN communities. Soil physico-chemicals properties, are known to be important factors for PPNs as they may modify their habitat, metabolisms, or movement for example [16,28] that could explain the highlighted effect of soil texture or rainfall. Heavy metals in soil such as Zn or Cu can shape PPN communities as these substances appeared to differentially decreased the abundance of PPNs in soil [14] as observed in this study for Pratylenchus, Trichodorus, Meloidogyne and Telotyenchidae, shown to be negatively correlated to Zn concentrations. The toxicity of heavy metals has been studied on growing media in Caenorhabditis elegans, a free-living nematode, and high concentrations have been shown to drastically increase mortality in both adult and juveniles [29,30], but also to reduce the growth of the nematodes, particularly in the descendants of parents exposed to heavy metals [30]. Heavy metals also alter the movement behaviour of the intoxicated individuals, possibly by attacking the muscles and the neural network [30]. At the community level studied here, little information is available about PPNs but heavy metals have an adverse impact on free-living soil nematode communities, thus including PPNs except cyst nematodes, by decreasing population abundances and community richness and modifying community structure [31,32]. Consequently, nematodes have developed a broad range of behavioural and physiological mechanisms (summarised in Ekschmitt and Korthals [14]) enabling avoidance, tolerance or detoxification of various heavy metals. The effects of heavy metals highlighted in this study can also be indirect through reduced plant growth [33] and thus lower quality nutritional content for PPNs, as these organisms depend on their host plants for nutrition.

Soil texture that appear in Fig 1 have also been shown in the literature to modify PPN community structure depending on the type of soil and particle size as they modify communities abundance and richness [13,16]. Sand, clay and silt proportions may affect the water retention but also the amount of minerals and organic matter present in soils [16,34]. This will impact plant growth and thus food quality for PPNs, but also movements through the water film around the soil particles, stimulated by the retention of root exudates that enable nematode to locate the roots [35], explaining the impact of soil texture on the communities sampled for this study.

Obviously, climate was a significant macro-variable affecting the PPN communities studied here, especially rainfall. Even at a larger scale (worldwide) Nielsen et al. [36] also observed an impact of rainfall on PPN communities. According to their observations, rainfall appears to be the main factor affecting nematode family composition (including non-PPNs) by influencing local soil properties and vegetation that drive the nematode communities’ structure. Temperature appears to be more important for PPNs in particular that appear the most abundant in warm locations, probably because of more stable ecosystems and abundant food resources than in cold location [36]. It is possible that in our study, soil variables were more important than climate compared to a worldwide scale, as climate variations in France, according to the world map of the Köppen-Geiger climate classification, are low and all the fields of this study were sampled in temperate regions with warm summers [37]. Precipitation, and thus moisture, increases plant growth, facilitates PPN movement in soil [38], and favours the PPN life cycle, for instance by increasing egg hatching [39].

Effect of field surface area on the PPNs is difficult to explain and could be an artefact. The sampling protocol were not fully adapted to the surface sampled (i.e. we did not sample more samples in the larger fields), but sampled seven soil cores over the longest diagonal in all fields. It therefore seems unlikely that the surface area affected PPN communities. More probably, field surface area reflects a characteristic of geographical regions as largest fields were sampled in Midi-Pyrénées and smallest fields were sampled in Nord-Pas-de-Calais.

Certain cultivation practices (e.g. tillage practice and application of non-herbicide products) also seem to play a key role for the PPN community’s structure in the fields, even though these practices did not impact the abundance of the PPN taxa individually. Profound tillage, for example, may impact food accessibility for PPNs, by removing weeds, as well as their living habitats, mainly their living depth and the soil texture, as individuals are moved deeply in the soil [40,41]. Furthermore, some PPNs are less sensitive to environmental changes as reported for Paratylenchus [24] and thus may benefit from the soil disturbance compared to the other PPNs. Non-herbicide plant protection products (i.e. insecticides, fungicides or molluscicides) may eliminate potential natural enemies of PPNs such as nematophagous fungi [42], but they also have a negative impact on non-target organisms such as the PPNs [43], explaining their structuring role on PPN communities.

Impact of the variables on each PPN taxon

Effects of the cultural practices and environmental variables were also tested on each PPN taxa using GLMMs. In our data, crop rotations were not retained in the GLMMs analyses after the dataset description using MCAs. This contrasts with results in the literature for which crops are known to be important for PPN community structure as richness and abundance vary with the crop rotations [44,45]. In our study, the impact of crop rotations may be masked by the environmental heterogeneity as at this country scale, environmental conditions (mainly soil) are much more diverse than the cultivation practices between the regions. Furthermore, and despite the presence of a root crop in the rotations, the dominance of cereals in all sampled fields, regardless of the region, probably homogenised the PPN communities.

We observed that herbicides have a significant negative impact, at least on long-term use, on the abundance of several PPN taxa (Pratylenchus, Trichodorus, Helicotylenchus, Paratylenchus, Criconemoides, Heterodera or Telotylenchidae), as has been reported previously [21,46,47]. It is not possible to rule out a potential direct effect of the products depending on the dose used [48], but it is more likely to be an indirect effect by removal of potential host weeds. Positive effects of herbicides can be observed on Telotylenchidae for accumulation of applications from years n-1 to n-4 (Table 2), possibly because of a negative impact on the other PPNs. Unidentified species among this family could be more competitive on the few remaining weeds. However, this interpretation is speculative, as the weed compositions, as well as potential intercrops between each year, were not assessed here.

In this study, soil physico-chemical variables (pH, soil C/N ratio and Zn) appears to be the main drivers of the PPN abundance variations for the identified taxa. The C/N ratio reflects organic matter and nitrogen enrichment. These variables generally characterise fertilizers to increase yields. As such, a positive effect may due to more dense root systems that are more convenient for PPNs development in the most fertilized fields. However, high nitrogen inputs for example have also been shown to be deleterious for soil nematodes because of the nematicidal effect of ammonia [15,49]. This was observed here because in the fields sampled for this study, the C/N ratios appear to be relatively low (<10)–except in the four fields sampled in Languedoc-Roussillon–and PPN abundances appear to be lower in soil with lower C/N ratios. With our dataset, Zn appears to negatively impact the abundance of Trichodorus, Meloidogyne, Pratylenchus and Telotylenchidae, confirming the results observed with the tb-RDA analyses for the whole PPN community.

Conclusions

Based on our results at the spatial scale of several regions of France, it appears that soil and climate conditions are more important than cultivation practices for PPNs abundance, and these conditions may mask the effects of certain cultivation practices. Heavy metals, soil pH, and rainfall deeply modify PPN communities, affecting their environment and access to—or quality of—food resources. We showed, with 35 sampled fields in eight different geographical regions, that local climate and soil variations, but also certain cultivation practices, could explain the PPN community variations, despite similar diversity. However, it should be emphasized that anthropogenic activities, mainly in agrosystems, completely change the soil conditions, with high inputs, or tillage practice, which directly or indirectly have an impact on PPNs.

Even in the similar agrosystems sampled here, PPN communities may vary and it is probable that the differences in diversity would be higher in more different crop rotations. In this case, we can hypothesise that PPN communities in the different agrosystems would not have an equal resilience capacity to introduction of a new species, such as Meloidogyne chitwoodi, and an equal capacity to limit its development. It has been shown previously in greenhouse experiments that more diverse communities, especially those with high overall abundance of PPNs, regardless of the species, are able to limit the establishment of M. chitwoodi [9]. Given these results, it seems important to consider herbicide use, nitrogen inputs, and soil physico-chemical composition, as well as the cultivated crop, to select the fields sampled for the French national monitoring programme for the Columbia root-knot nematode. Further investigations are also needed to assess the potential of the different natural communities, which here do not damage crops, to control and limit the development of frequently introduced species, with the aim of proposing alternatives to pesticide use.

Methods

Ethics statement

No specific permits were required for the samplings. Permissions for sampling the fields were granted by the farmers (landowners). Furthermore, the 35 fields sampled in this study were also part of the 2015 French national monitoring programme for the Columbia root-knot nematode. The lands are not protected in any way and no protected plant or animal was sampled.

Sampling and characteristics of the studied areas

Sampling was carried out from June to September 2015 in the same fields as those sampled in the framework of the national monitoring program dedicated to Meloidogyne chitwoodi and M. fallax. For this program, fields are chosen randomly among those that have grown a susceptible crop to M. chitwoodi, mainly root crops such as carrots, potatoes or beets. Eight regions were investigated: Aquitaine, Brittany, Languedoc-Roussillon, Midi-Pyrénées, Nord-Pas-de-Calais, Upper and Lower Normandy and Picardy. Sampling was repeated in 2016 in the same fields except three fields in Brittany and one field in Nord-Pas-de-Calais for which farmers did not allow us to sample again. All these fields are present in geographical areas defined as temperate regions with warm summers [37]. Mean monthly temperatures and rainfall data were collected from the closest meteorological station from “Terre-net.fr”, an agricultural data website [50] monitoring climate in fields in France, for each sampled field over the year before the sampling date.

A total of 35 fields, ranging from 0.13 ha to 6.64 ha and representing 31 farms were sampled in 2015, and 31 of them were sampled again in 2016 as farmers from the other four fields (3 fields from Brittany and 1 field from Nord-Pas de Calais) did not complete the survey. For each field, information about cultivation practices from 2010 to 2016 was collected from farmers through a survey: crop rotations, type and number of tillage practices (profound tillage at 30 cm or deeper and superficial at less than 30 cm deep), and number of applications of plant protection products (herbicides or non-herbicides (including fungicides, insecticides and molluscicides). The physico-chemical properties of soils, including pH, organic C/N ratio, soil texture and Cu, Zn and Fe quantities, were obtained from “BDAT”, a public database of the French National Institute for Agricultural Research and Environment [51]. Soil samples (around 1.5 kg) were composed of seven elementary soil cores taken at 30 cm depth along the longest diagonal of each field with a manual auger (diameter 2.5 cm). The GPS coordinates were recorded at each of the seven sample points in 2015 in order to repeat the same sampling in 2016.

Nematode extraction and identification

The extraction protocol was the same as in Garcia et al. [21]. Briefly, mobile stages were extracted from 300 mL of fresh soil according to EPPO protocol for nematode extraction [52], using an Oostenbrink elutriator (Meku, Germany). Extraction was followed by two centrifugations (Hettich Rotanta 460, Germany), the first one with water and the second one with MgSO4 with a density of 1.18 [52], to purify the nematode suspension. PPN genera were identified and counted in 5 mL of the suspension after a dilution step depending on the quantity of nematodes in the total suspension. Identifications were based on morphological criteria using a stereomicroscope or a microscope when more precision was needed [53,54]. Cysts were manually isolated from 500 mL of fresh soil under stereomicroscope after an adapted Baunacke methods on two successive sieves (800μm and 170μm) [52]. Genera were identified based on cyst morphology. Furthermore, the number of cysts was transformed into the number of eggs and juveniles per cyst, through extrapolation after crushing thirty-three cysts, to enable abundance comparisons with the other PPN taxa.

Statistical analysis

For this study, we considered 15 different variables, both quantitative and qualitative: year of sampling, mean monthly rainfall, mean monthly temperature, C/N ratio, soil texture, soil Cu, Zn and Fe concentrations, soil pH, field surface area, crop rotation, number of superficial and profound tillage practices, number of herbicide and non-herbicide products used.

All statistical analyses were conducted using R software [55]. The Shannon index of species diversity was calculated for each PPN community as detailed in Spellerberg and Fedor (2003) [56]. A Wilcoxon test was used to assess differences between PPN taxa abundance and Shannon index in 2015 and 2016.

As samples were taken in quite different areas (mainly regarding environmental conditions), we wanted to assess the impact of both cultivation and environmental variables on the whole PPN community. Transformation-based redundancy analysis (tb-RDA) was performed using the “Vegan” R-package [57]. Since our dataset contained many zeros and according to Legendre and Gallagher (2001) [58] and Legendre and Legendre (2012) [59], we transformed our community data using Hellinger transformation [60] to allow the use of RDA (that is based on Euclidean distance, very sensitive to community matrices containing many zeros). Furthermore, the significance of the most contributive variables, was tested according to the Akaike information criterion (AIC) after both forward and backward selection with permutation tests. We performed six tb-RDAs to assess the impact of the past cultivation practices on the present PPN communities. For each analysis, the cultivation practices of the previous years were implemented one by one, moving backward (e.g. practices of year n for the first tb-RDA, practices of years n and n-1 for the second, practices of the years n, n-1 and n-2 for the third etc.), in order to evaluate whether accumulation of tillage practice or pesticides over the years could reveal a cumulative effect.

The effects of the variables on the abundance variation of each PPN taxon were evaluated, using a statistical strategy similar to that described by Garcia et al. [21]. Briefly, we first used Multiple Correspondence Analysis (MCA) using the “FactoMineR” R-package [61] to describe our dataset and to select the main contributing variables without any a priori knowledge [62,63]. MCA enables implementation of both qualitative and quantitative variables after grouping the values of the quantitative variables into two to four classes, in a way as balanced as possible to avoid giving greater weight to one modality compared to the others. Limits of each classes were the first and third quartiles and the median of the distribution of each variable. The PPN abundances were considered as supplementary variables and only the most contributive classes or modalities of variables (with an absolute contribution higher than twice the mean absolute contribution [64,65]) were kept on the factorial maps. Six MCA analyses were also performed, adding for each, the cultivation practices of the past years similarly to the tb-RDAs. We retained the variables for which two modalities at least were present on not less than two factorial maps. Then, the effective relationships between these selected variables and the abundance of the PPN taxa were tested using a model-averaging approach through the corrected Akaike information criterion (AICc) [62,63] on generalised linear mixed Poisson models (GLMMs) [66]. We used the “lme4” R-package [67] and the “MuMIn” R-package [68]. We considered the field surface area as a random variable since the sampling protocol was not adapted to the field surface area. According to Grueber et al. [63], we transformed our explanatory variables using Gelman’s (2008) [69] approach, using the “arm” R-package [70], when the models failed to converge. The Sum of Weight (SW) and 95% Confident Interval (CI) were calculated for the variables present in the subset of models with a ΔAICc < 6. Furthermore, if the 95% CIs included zero, the effect of the variables was considered uncertain [63] and thus not kept for the final model.

Outputs of the tb-RDAs for each year period of cultivation practices.

Arrows and bold texts indicate the significant variables after a permutation test. Modalities of soils depend on the proportion of sand, silt and clay in it (SoilSaSiC = majority of sand, then silt then clay; SoilSiCSa = majority of silt, than clay, then sand; SoilSiSaC = majority of silt, then sand, then clay)A: cultivation practices of year n and n-1 are implemented, the first two axes presented explain 38% of the dataset variance; B: cultivation practices of the n to n-2 are implemented, the first two axes presented explain 36% of the dataset variance; C: cultivation practices of year n to n-3 are implemented, the first two axes presented explain 35% of the dataset variance; D: cultivation practices of year n to n-4 are implemented, the first two axes presented explain 39% of the dataset variance; E: cultivation practices of year n to n-5 are implemented, the first two axes presented explain 36% of the dataset variance.

(TIF)

Click here for additional data file.

Outputs of the MCAs for each year period of cultivation practices.

Only the most contributing variables are projected on the maps. PPNs do not contribute to the factorial map construction. Classes for each variable are based on first quartile, median and third quartile. Abbreviations are presented in the supplementary material S2 Table. A: only cultivation practices of year n (year of sampling) are implemented in the analysis; B: cultivation practices of year n and n-1 are implemented; C: cultivation practices of year n to n-2 are implemented; D: cultivation practices of year n to n-3 are implemented; E: cultivation practices of year n to n-4 are implemented; F: cultivation practices of year n to n-5 are implemented.

(TIF)

Click here for additional data file.

Raw model-averaging results for each taxon and year periods.

For each taxon, variable and year period considered, estimate, after the model averaging approach, 95% confident interval (95% CI) and the sum of weight (Σωi) are presented for the variables retain after the selection step.

(XLSX)

Click here for additional data file.

Detail of the abbreviations used in MCA analysis outputs.

(XLSX)

Click here for additional data file.

Complete dataset (PPN community and environmental and cultural variables) for each year of sampling.

(XLSX)

Click here for additional data file.

23 Jul 2021

PONE-D-21-19128

Diversity of plant parasitic nematodes characterized from fields of the French national monitoring programme for the Columbia root-knot nematode

PLOS ONE

Dear Dr. Garcia,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

I appreciate your objective to explore the significance of various biotic and abiotic parameters for the distribution of plant parasitic nematode taxa, as we definitely lack understanding on this matter. That said, both reviewers raise a number of critical points, to which I agree. In its present form, your manuscript is not acceptable for publication, and I will only be able to assess its quality after substantial revision, where you carefully address all comments and points raised by the reviewers.

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Academic Editor

PLOS ONE

Additional Editor Comments:

I appreciate your objective to explore the significance of various biotic and abiotic parameters for the distribution of plant parasitic nematode taxa, as we definitely lack understanding on this matter.

That said, both reviewers raise a number of critical points, to which I agree. In its present form, your manuscript is not acceptable for publication, and I will only be able to assess its quality after substantial revision, where you carefully address all comments and points raised by the reviewers.

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere. [This research article is linked to the 2nd chapter of my PhD thesis, defended at the end of 2017, available on the HAL open archive (it is mandatory for French Ph.D. students), but never published in a peer-reviewed journal. The data, the analyses and the results presented are not strictly the same as those from my Ph.D. thesis manuscript and majority of the text has been reformulated.] Please clarify whether this [conference proceeding or publication] was peer-reviewed and formally published. If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript describes the effect of biotic and abiotic parameters on plant parasitic nematode communities and raises the question, if certain parameters can be used in the future to select "high-risk" fields for the detection of certain quarantine nematodes in monitoring programs. Unfortunatley, this hypothesis could not be addressed as no quarantine nematodes were detected. Besides, in praxis it is much easier and straight-forward to select fields based on good host plants, so the benefit of using plant parasitic nematode communities is not clear. If nematode communites are used to characterize certain scenarious, non-plant parasitic nematodes play an important role. However, this group of nematodes was completley ignored in the present study. It should at least be mentioned why non-plant parasitic nematodes were not considered in the study. Besides, there are numerous shortcomings and missing information as indicated in the separate file. Finding approriate answers to the raised question will decide, if the manuscript will finally be acceptabe or not.

Reviewer #2: NOTES FOR THE AUTHORS:

This is a novel study, and potentially a good addition to the literature that characterizes the ecology of plant-parasitic nematodes, however I would recommend publication only if major revisions are undertaken. The presentation of the manuscript is not yet of publication quality either by the standards in the field or by the expectations of the journal. Arguments and discussion are not fully developed, nor are they detailed, and frequently the reader is left attempting to follow a line of reasoning that relies entirely upon a necessity to read the cited references. There is heavy use of vague and/or subjective language (“various”, “several”, “important factors”, “major”, “more or less”, “certain groups”, “few”, “seems to vary”, “numerous”, “better quality”, etc.) which requires further explanation. Subjective language is used to describe changes or degrees of effect; this should be replaced with quantitative values or neutral language. Furthermore, the modelling and conclusions in this study are heavily reliant upon soil metadata that was not measured for this study, but retrieved from a database, potentially rendering these conclusions invalid if the metadata has changed.

INTRODUCTION:

General note:

Frequently, explanations and lines of argument are neither clear nor detailed, and often the reader is left attempting to follow a line of reasoning that has large gaps. For example, if a statement is made that cultivation practices or environmental conditions affect nematodes, this statement needs to be followed with explanatory text describing the specific types of disturbances that affect nematodes, and what the effects on the nematodes or nematode populations are.

Line 44: Technically unicellular organisms are considered to be micro-organisms, whereas nematodes (multicellular) are considered microfauna or mesofauna. Please adjust this wording for clarity and consistency with the field.

Line 47: Does “natural population levels” refer to crop population abundances or nematode abundances? Please specify.

Lines 55-57: “…could be potential antagonists.” This sentence is unclear, and the implication is vague; please specify what the introduced species could be antagonists for or to, and how the antagonism might occur.

Line 70: The term “soil work” is a non-standard term that is used frequently throughout this manuscript in reference to cultivation practices. In the interests of clarity, please use the standard terms “tillage practices” or “cultivation practices”, and further detail as necessary.

Line 72: Please describe how cultivation and physico-chemical properties of the soil affects nematodes. When creating a line of reasoning it is not sufficient to simply state that there is an effect, the effect should be described.

Line 73: Please list types of environmental conditions and crop practices that have been studied.

Lines 73-74: Please describe the nature of restricted areas and limited experimental designs.

Line 78: Please remind us of the importance of a root crop vs. a cereal crop.

RESULTS:

Lines 104, 106: Please specify whether per 100 g soil refers to 100 g wet (or field-moist) soil or dry soil?

Lines 107-109: I did not see any information in the Methods about performing calculations for the Shannon Index. Please include methodology, reference(s), and data.

Lines 110-112. This sentence is unclear. What does this mean?: “…and Meloidogyne sp. mentioned in the article correspond to other species.”

Line 136: Misspelling of “this”. “…cumulative effect of few practices…”; please list the practices to which you refer. Also, it is mentioned that agricultural practices were not retained in the analysis, and the sentence immediately contradicts itself by stating that effects due to practices were observed. Could this entire sentence be clarified?

Line 138: Please specify what you mean by a field surface. Whether or not a result is difficult to explain it should be listed in the results; further explanation can be done in the discussion, and if indeed the explanation is due to analysis artifacts, further explanation of the nature of the artifact is needed.

Lines 139-140: The methodology as described in this sentence appears to match sampling protocols outlined in the Methods section, thus contradicting the opening of the sentence. Please describe in detail what was not adaptable for this particular sampling.

Line 143: Please reference the appropriate figure or table after “…factorial map.”

Line 144: “seem” is subjective; simply list the impact of the variables quantitatively or statistically rather than rendering a judgment call as to perceived importance. Commentary of that nature should be moved to the discussion.

Line 144: By “structuration” do you mean “community structure”? Structuration is not the standardly accepted term for community structure, nor is it commonly used to refer to the formation or change of community structure.

Lines 167-182: The definitive statements “None of these variables impacted…”, “…had a positive impact…”, “…had a negative impact…” cannot be made. Remember that this study is largely statistical, and that that metadata originates from soil data that has not been measured in the laboratory for this study. At most it is possible to state that “…have appeared to positively/negatively impact…”. This paragraph requires significant clarification.

DISCUSSION:

General: There is frequent use of vague and subjective language (“various”, “several”, “seems”, “important factors”, “major”, “more or less”, “certain groups”, etc.) throughout this section. Please remove all subjective language and reframe in terms of quantitative and/or statistical values. Vague terminology requires clarification in the text, rather than following with a citation so that the audience is required to read the citation in order to draw their own conclusions.

Lines 187-188: “Various” is vague usage. Please specify the geographical areas and crop management approaches to which you refer.

Line 189: What do you mean by “close to that of other similar agrosystems”? “Close” is subjective; what of your data supports this assertion, and please be very specific in your response. What are “similar” agrosystems?

Lines 194-195: “Majority of taxa” is undefined. Please specify percent or number of taxa. “Several regions” is undefined. Specify how many regions, which ones they are, and discuss the importance of these regions with respect to crops, nematodes, etc. “Seems to vary” is undefined and subjective. Specify numerically or statistically the degree to which taxa vary, and what they vary with respect to (presence, abundance, etc).

Line 203: Please specify what economic issues are induced.

Line 204: Rather than “One of these two crops…”, crops should be specified.

Line 214: This entire sentence is vague. Specify soil conditions to which you refer, and how these collective soil conditions and/or abiotic factors influence PPN and free-living nematodes. Discuss how these collective soil and abiotic factors stimulate or repress nematode growth and development, and why this is important to your argument.

Line 216: “More or less susceptible” is vague. Explain how nematodes are susceptible to the presence of Zn and Cu in (presumably) soil.

Line 220: Do you mean “intoxicated” or nematodes that suffer from toxic effects?

Lines 222-223: Specify the “strong impact” due to heavy metals.

Line 228: “Major” is subjective; simply list the impact of soil texture on community structure quantitatively or statistically rather than rendering a judgment call as to perceived importance. Is the effect of statistical significance? Usage of “structuration” again.

Line 231: “Eased” is subjective. Please clarify.

Lines 235-237: Specify how rainfall affects nematode communities, how temperature affects PPNs, and why other soil variables are not considered important (and specify the soil variables). Why are all of these effects the case?

Line 239: Sentence appears to contradict itself, can this be made clearer?

Lines 242-243: “Soil work” and “structuration”—see notes above.

Lines 253-260: I’m having trouble following the flow of the argument; please clarify. Also, on line 256, the statement is made that the study has a large geographic scale, whereas in line 239 the opposite is stated. Please clarify.

Line 266: “certain groups of years”. Specify the dates and/or date ranges.

Line 271-272: “Their effects appeared to be positive or negative, probably depending on the PPN ecological niche.” This sentence is vague and requires considerable expansion; please discuss the specific positive and negative effects, and how these effects are or could be driven by a given ecological niche.

Line 273: “inputs”; please specify the inputs that you are referring to and how inputs are characterized to increase yield.

Line 274: “better quality” is subjective. What, specifically, constitutes quality, a change in quality, and what do these changes induce in nematode consortia?

CONCLUSIONS:

Lines 285, 287: In the discussion crop rotations are described as having been removed from analyses, therefore it should not be possible to draw conclusions from them. Please clarify.

Line 289: By “population” do you mean “abundance”?

MATERIALS AND METHODS:

Lines 317-318, 320: It is safe to say that many readers will not be familiar with the national monitoring program. Within the regions listed, please specify field site locations, including the geolocations of the sites. In line 320, specify which regions, sites, and fields were used and note where changes occurred between years.

Line 322: Specify the name of the agricultural data website, not just the reference. Were mean temperatures obtained for each field individually? If not, how were temperatures determined?

Line 333: What is an “elementary soil core”? This is not terminology that I am familiar with.

Line 344: Should this be “sieves”? If so, please specify all sieve sizes as well as any standard protocols used.

Line 345-346: “based on personal observations” is vague; specify the exact methodology or calculation required to determine the number of larvae per cyst.

Line 351: Please define “field surface”.

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: PONE-D-21-19128 comments.docx

Click here for additional data file.

3 Nov 2021

Response to the points raised by the academic editor and reviewers

Dear editor,

Please find here a response to reviewers’ comments and a substantially revised manuscript according to all the points raised. According to numerous comments from both reviewers, many sentences have been modified to remove subjective language, and clarifications have been made for table 1 and Methods section. As reviewers pointed out some misuse of different terms, supporting informations have also been modified to match the vocabulary in the manuscript.

Sincerely

Dr. Nathan Garcia

Editor comments:

• Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

_ Style and format have been checked and modified through the whole manuscript to meet PlosOne’s style requirements according to the templates from the links. Fig 1 has been uploaded to the PACE digital diagnostic tool as recommended.

• Please clarify whether this [conference proceeding or publication] was peer-reviewed and formally published. If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript.

_ This work was not peer-reviewed nor formally published. In fact, some of the results were obtained and presented in my PhD thesis, defended at the end of 2017, and the whole thesis manuscript is available on the HAL open archive (which is mandatory for French Ph.D. students), without any copyrights. Co-authors and I have continued working on this study since my PhD defense and thus the analyses and the results presented in this manuscript are not strictly the same as those from my Ph.D. thesis manuscript and most of the text has been rewritten. There is no reason to consider this submission as a dual publication of my PhD thesis manuscript.

Reviewer #1 comments:

• Nematodes are not microorganisms, they belong to the mesofauna, please correct

_ Reviewer #2 also raised this point. The sentence has been removed from the abstract and corrected latter in the manuscript.

• To compete is not enough, they need to suppress the non-native species. Rephrase.

_ Ideally reviewer #1 is right, however we have few evidences of such suppression as in Garcia et al., 2018 (DOI: 10.1111/ppa.12914) we observed suppression of M. chitwoodi in only 5 pots out of 80 after a 4 months experiment. But abundance decrease has been observed much more frequently and we can expect that over a longer period this can lead to suppression or at least keep the non-native species at a non-damageable level. So to suppress is indeed the best solution but to compete is already a quite interesting observation as it can lead to suppression or some control over time.

• “…using non-damaging indigenous species…” applies artificial application of those specimen, which is not realistic. I think what is meant is, that non-damaging species should be supported in order to suppress damaging species.

_ It is indeed what we meant. The sentence has been modified accordingly.

• Regarding the target organisms, I don’t think we can differ between non-native and native damaging species. Here we need a more general approach like “suppressing damaging species including non-native species.” Or similar.

_ We agree if the non-native species is already established in the soil. However, we placed this work in a scheme of an introduction of a species listed as quarantine organism that is not currently widely distributed in Europe and thus we consider that supporting indigenous non-damaging species may decrease the abundance of such introduced species.

• Looking of the spectrum of identified genera, they belong to more than one family. Rephrase accordingly.

_ Sentence has been rephrased. We identified 10 plant-parasitic nematode taxa, and for one of them, we were not able to identify the genus but only the family.

• Soils are part of fields. No need to mention separately.

_ Sentence has been modified. By “field” we meant “cultural practices” and by “soil” we meant “soil physico-chemical properties”.

• I don’t think data were not collected over six years, but instead field data representing the past six years were collected. Clarify.

_ Sentence has been modified. Indeed, we meant “representing the past six years”.

• … than what? Second partner of comparison needs to be mentioned.

_Sentence has been completed.

• Both aspects were not part of the study. Food quality refers to crop plant species grown and soil food web to the entire soil ecosystem. The presented data do not provide any information on the food quality of the crops grown nor on the characterization of the soil food web (suppressiveness etc). Change accordingly.

_ Sentence has been removed. We agree that the sentence was too general compared to the data analyzed in this work.

• Uncommen wording within this context. Do you mean establishment or spreading? Why managing and not controlling/eradicating/suppressing the introduced nematode? Rephrase accordingly.

_ “implantation” has been replaced by “establishment” all over the manuscript. This point was also raised by reviewer #2. To our opinion, “manage” is synonym to “controlling” suggested by reviewer #1 and as mentioned in a previous comment, we have only few evidence that “eradicating/suppressing” is possible, while abundance decrease is more obvious and already a useful lever.

• Need to be mentioned, how this can be done. This is the most relevant aspect according to the authors, and thus the procedure should be described in detail. What are the options to promote diversity? How does the information gained from the collected data specifically help to promote diversity. Give at least suggestions.

_ Sentence has been removed since the reasoning was too indirect for the abstract for which we do not have space for more details. Here we meant that the variables highlighted in our work could positively influence PPN communities’ richness and abundance and thus promote biodiversity.

• See previous comment

_ “micro-organisms” replaced by “mesofauna”.

• No need to mention

_ Agree, text has been removed.

• What are natural population levels? Needs explanation.

_ Reviewer #2 also raised this point. “population level” has been modified by “abundance” all over the manuscript. By “natural abundance” we meant the abundance that is commonly observed in French fields, both according to our expertise but also to published data like Villenave et al. (2013) (DOI: 10.4236/ojss.2013.31005) in which they sampled and analyzed the nematode communities from 52 fields or Garcia et al. (2018) (DOI: 10.1163/15685411-00003136) where 72 fields were considered.

• establishment?

_ Yes, word has been replaced.

• See previous comment

_ Same.

• What do you mean by “competition between communities”? specify.

_ Sentence has been rephrased.

• I guess it is more the composition than the structure

_ As reviewer #2 suggested, we have replaced “structuration” by “community structure” for the whole manuscript.

• It is not clear to the reader how that information will improve national monitoring programs. A good understanding of the diversity and composition of nematode communities is highly complex and time consuming. I doubt that it will be realistic in the future to select fields in monitoring programs based on nematode diversity and composition. The main and most likely only factor is host plant status. Rephrase accordingly.

• As mentioned above, if you want to find M. chitwoodi, you sample good host plants at the end of the season (highest expected numbers) considering possible ways of entrance (e.g. fields were seed potatoes were planted). Composition of plant-parasitic nematodes is not an issue and I doubt it will be in the future.

_ Sentences pointed out by these two comments has been modified to make our reasoning clear. We totally agree that for national monitoring program for M. chitwoodi, it is essential to sample fields based on good host plants and we are not questioning this in the study. However, it is not humanly possible for the sanitary services to sample every fields with a good host plant in the country. Thus, we wanted with this work to promote additional elements that can be considered to choose in the most efficient way the fields to sample among those with a good host plant. As indigenous plant-parasitic nematode communities may decrease M. chitwoodi abundance, and as soil properties, cultural practices and climate may impact plant-parasitic nematode communities, we believe those should be relevant elements to consider in addition to the host plant.

• Tillage per se can also enhance plant-parasitic nematode densities, such as minimum tillage. Thus it is more the intensive tillage including ploughing that has a negative impact on ppn. Please specify.

_ Sentence has been modified.

• “soil” indicates all nematodes, but non-plant parasitic nematodes are not covered in this study

_ Agree, the modification proposed by reviewer #1 has been accepted.

• Those numbers are quite high. Is this really juveniles or could it be cyst content (eggs + juveniles)?

_ It is an extrapolation of Heterodera cyst content.

More details have been added in Methods section according to a latter comment from reviewer #1 and a comment from reviewer #2

In a previous work published by Garcia et al. 2018 in Nematology (DOI 10.1163/15685411-00003136) we investigated the number of juveniles and eggs in Heterodera cysts sampled in 7 fields in Burgundy (France) during two consecutive years. A total of 33 cysts belonging to different species were individually crushed and the number of juveniles and eggs counted. The obtained mean juveniles + eggs per cyst was then used to convert cyst numbers of other French fields into juvenile and eggs numbers.

• See comment above

_ We removed “micro” as suggested

• composition?

_ “Structuration” has been replaced by “community structure” as suggested by reviewer #2

• Composition?

_ See previous comments.

• It is not obvious how this can be the case and thus needs explanation.

_ Sentence has been more detailed. We meant here that profound tillage modifies soil texture, destroy weeds and move the nematodes deeply in the soil. Since these tiny organisms have limited active migration abilities, profound tillage negatively affects food accessibility.

• Composition?

_ See previous comments.

• What kind of groups? Needs explanation

_ We detailed here the group of years mentioned in Table 2.

• Or alternatively, ….were lowest in soil with the lowest C/N ratio.

_ The suggested changes have been accepted.

• Pesticides in general? The survey only mentions herbicide. Stick to what has been studied and avoid generalizing things.

_ Sentence containing this term has been removed (see following comments from reviewer #2). However, to answer the question, here we meant pesticides in general (including herbicides). In the survey, farmers have indicated the number of herbicide application and other pesticides application (meaning fungicides, molluscicides and insecticides) that we called “non-herbicides” in the manuscript. As farmers are not always prone to give details about the other pesticides applications, we have had to stick to the sole total amount of non-herbicides application.

• are soil work, tillage and cultivation practices used synomously? If so, stick to one term (cultivation practices) and use this consistently. Change in entire text.

_ In the manuscript “cultivation practices” is more general and contain tillage practices, herbicide and non-herbicide applications and crop rotations.

Furthermore, according to reviewer #2 comment, “soil work” have been replace by “tillage practices” in the entire manuscript and we have detailed “superficial” or “profound” each time.

• Mention the criteria for the selection of those fields

_ Details about the selection of the fields in the French monitoring program have been added.

• Too general as not all heavy metals were determined. Specifically mention those that have been considered in this study.

_ “Heavy metals” have been replaced by those considered in this work.

• I don’t think there are techniques that distinguish between free-living and cyst nematodes. But there are techniques specifically for mobile stages and cyst stages. Is that what is meant? Needs clarification and precise wording.

_ Yes, we meant “mobile stages”, “free-living” has been replaced.

• Using the same technique, i.e. Oostenbrink elutriator? This does not make sense. Describe more preciously how extraction was done and mention a reference

_ The whole extraction paragraph has been substantially more detailed, according to the appropriate reference, the EPPO extraction protocols for nematodes.

• Two times the same centrifugation? Needs explanation.

_ More details have been added. The first centrifugation is done with water and the second with MgSO4 with a density of 1.18.

• See previous comment

_ “free-living” has been removed from the modified sentence.

• I thought the nematodes are already extracted at this step. So, where does the soil extract comes from? Do you mean nematode suspension? Usually, nematodes are concentrated in a certain volume (e.g. 10 ml) and then an aliquot (e.g. 1 ml) is used to identify and count the specimen. Preciously describe the method used.

_ Yes we meant “nematode suspension”, the words have been replaced. More details about the dilution step have also been added. We counted the PPNs from 5mL of a diluted suspension (usually 100mL) depending of the abundance of nematode in the total suspension to be able to correctly identify and count them.

• …. or eggs plus juveniles per cyst? (= cyst content)

_ Yes we meant juveniles and eggs. Sentence has been modified accordingly.

• Difficult to understand how this was done. Generally, a defined number of cysts is crushed and cyst content in terms of number of eggs and juveniles determined and calculated back according to 100 g soil.

_ See previous comments for the detailed method. Information has been added.

• What is the message of Legendre and Gallagher? Explain

_ Details about the cited reference have been added at the end of the sentence.

• The number of references is far too long for a research article of this type. Keep in mind that this is not a review article. Condense number of references to 30-40. This can be easily done by focusing on the most relevant references and/or referring to review articles regarding general aspects.

_ We have reduced the number of cited reference by 13 but we preferred and strongly believe that it is best practice to refer to original publications than to reviews citing these original publications. Moreover, PlosOne guide for author does not limit the number of references.

Reviewer #2 comments:

• Line 44: Technically unicellular organisms are considered to be micro-organisms, whereas nematodes (multicellular) are considered microfauna or mesofauna. Please adjust this wording for clarity and consistency with the field.

_ See comment from reviewer #1.

• Line 47: Does “natural population levels” refer to crop population abundances or nematode abundances? Please specify.

_ “Population level” has been replaced by “abundance” in the whole manuscript according to reviewer comment.

• Lines 55-57: “…could be potential antagonists.” This sentence is unclear, and the implication is vague; please specify what the introduced species could be antagonists for or to, and how the antagonism might occur.

_ Sentence has been rephrased and an example of antagonism between indigenous PPN communities and introduced species is given in the following sentence.

• Line 70: The term “soil work” is a non-standard term that is used frequently throughout this manuscript in reference to cultivation practices. In the interests of clarity, please use the standard terms “tillage practices” or “cultivation practices”, and further detail as necessary.

_ “Soil work” has been modified in the manuscript and figures accordingly to this comment.

• Line 72: Please describe how cultivation and physico-chemical properties of the soil affects nematodes. When creating a line of reasoning it is not sufficient to simply state that there is an effect, the effect should be described.

_ Details have been added to the sentence.

• Line 73: Please list types of environmental conditions and crop practices that have been studied.

_ Sentence has been completed.

• Lines 73-74: Please describe the nature of restricted areas and limited experimental designs.

_ Sentence has been removed to avoid repetition with the previous and following ones.

• Line 78: Please remind us of the importance of a root crop vs. a cereal crop.

_ Details have been added and the beginning of the Methods section has also been more detailed according to a latter point raised by reviewer #2.

• Lines 104, 106: Please specify whether per 100 g soil refers to 100 g wet (or field-moist) soil or dry soil?

_ We meant 100g of fresh soil. This information has been added.

• Lines 107-109: I did not see any information in the Methods about performing calculations for the Shannon Index. Please include methodology, reference(s), and data.

_ Those informations have been added to the Methods section. A line regarding Shannon index has been added to table 1.

• Lines 110-112. This sentence is unclear. What does this mean?: “…and Meloidogyne sp. mentioned in the article correspond to other species.”

_ In French legislation, it is mandatory to look for Meloidogyne chitwoodi and M. fallax if we found any Meloidogyne sp. individuals in a sample. As we found some Meloidogyne in several samples, we ensured that it was not one of the two quarantine species. Sentence has been modified to make it clear.

• Line 136: Misspelling of “this”. “…cumulative effect of few practices…”; please list the practices to which you refer. Also, it is mentioned that agricultural practices were not retained in the analysis, and the sentence immediately contradicts itself by stating that effects due to practices were observed. Could this entire sentence be clarified?

_ Sentence has been clarified. On Fig 1, only cultural practices of the sampling year are considered and none significantly affects the PPN communities (i.e. none appeared on the factorial map). However, when the cultural practices from the past years are also considered, effect of the successive non-herbicide applications and of the successive profound tillage appeared on the factorial maps presented in Fig S1.

• Line 138: Please specify what you mean by a field surface. Whether or not a result is difficult to explain it should be listed in the results; further explanation can be done in the discussion, and if indeed the explanation is due to analysis artifacts, further explanation of the nature of the artifact is needed.

_ Field surface is the field area, measured in hectare.

This result is still present in the Results section but we move the rest of the paragraph regarding a possible artefact in the Discussion section with clarifications of our reasoning (line 271-277 in the track changes version).

• Lines 139-140: The methodology as described in this sentence appears to match sampling protocols outlined in the Methods section, thus contradicting the opening of the sentence. Please describe in detail what was not adaptable for this particular sampling.

_ Here we meant that we did not sample more soil in largest fields. Sentence has been modified to clarify.

• Line 143: Please reference the appropriate figure or table after “…factorial map.”

_ Reference to the Fig 1 has been added.

• Line 144: “seem” is subjective; simply list the impact of the variables quantitatively or statistically rather than rendering a judgment call as to perceived importance. Commentary of that nature should be moved to the discussion.

_ Sentence has been modified to remove “seem to”.

• Line 144: By “structuration” do you mean “community structure”? Structuration is not the standardly accepted term for community structure, nor is it commonly used to refer to the formation or change of community structure.

_ Yes, we meant “community structure”. The word has been modified for the whole manuscript.

• Lines 167-182: The definitive statements “None of these variables impacted…”, “…had a positive impact…”, “…had a negative impact…” cannot be made. Remember that this study is largely statistical, and that that metadata originates from soil data that has not been measured in the laboratory for this study. At most it is possible to state that “…have appeared to positively/negatively impact…”. This paragraph requires significant clarification.

_ The whole paragraph has been modified to remove definitive statements about soil variables (lines 183-200 of the track changes version). We agree that the soil data were not measured during this work but we have minimized this bias as much as possible by using the database developed by the French National Research Institute for Agriculture, Food, and Environment. Data are available at the canton scale (the smallest French administrative scale with a mean surface of 192Km²). For each canton, more than 10 soil samples were analyzed over the canton surface between 2010 and 2014. As the samples for this work took place in 2015 and 2016, we considered that those data were close enough to our conditions.

• Lines 187-188: “Various” is vague usage. Please specify the geographical areas and crop management approaches to which you refer.

_ Sentence has been clarified. This first sentence was general to highlight that each field presented different environmental conditions (climatic and soil) and different cultural practices (for example from 0 to 6 profound tillage for the past 6 years).

• Line 189: What do you mean by “close to that of other similar agrosystems”? “Close” is subjective; what of your data supports this assertion, and please be very specific in your response. What are “similar” agrosystems?

_ Sentence has been clarified. We meant agrosystems that also include cereals and vegetables in the crop rotation similarly to the fields sampled in our study.

• Lines 194-195: “Majority of taxa” is undefined. Please specify percent or number of taxa. “Several regions” is undefined. Specify how many regions, which ones they are, and discuss the importance of these regions with respect to crops, nematodes, etc. “Seems to vary” is undefined and subjective. Specify numerically or statistically the degree to which taxa vary, and what they vary with respect to (presence, abundance, etc).

_ Sentence has been modified to give numbers of taxa and regions. We remove subjective end of the sentence, which is discussed in the following sentence.

• Line 203: Please specify what economic issues are induced.

_ We specify the economic issues from the cited references.

• Line 204: Rather than “One of these two crops…”, crops should be specified.

_ Crops (beet or carrot) mentioned in the previous sentence have been specified here.

• Line 214: This entire sentence is vague. Specify soil conditions to which you refer, and how these collective soil conditions and/or abiotic factors influence PPN and free-living nematodes. Discuss how these collective soil and abiotic factors stimulate or repress nematode growth and development, and why this is important to your argument.

_ Sentence has been modified to be more specific about the abiotic factors and the discussion about their impact is detailed during the rest of the paragraph.

• Line 216: “More or less susceptible” is vague. Explain how nematodes are susceptible to the presence of Zn and Cu in (presumably) soil.

_ Sentence have been detailed with information from the cited reference.

• Line 220: Do you mean “intoxicated” or nematodes that suffer from toxic effects?

_ Yes we meant “intoxicated”. Sentence has been slightly modified to make it more understandable.

• Lines 222-223: Specify the “strong impact” due to heavy metals.

_ Sentence has been clarified in order to develop the reasoning.

• Line 228: “Major” is subjective; simply list the impact of soil texture on community structure quantitatively or statistically rather than rendering a judgment call as to perceived importance. Is the effect of statistical significance? Usage of “structuration” again.

_ We removed the word “major” and added information about the kind of impact of soil texture. Furthermore, more details are given in the following sentences.

• Line 231: “Eased” is subjective. Please clarify.

_ Sentence has been clarified to explain that root exudate are used by nematodes to locate their host plants.

• Lines 235-237: Specify how rainfall affects nematode communities, how temperature affects PPNs, and why other soil variables are not considered important (and specify the soil variables). Why are all of these effects the case?

_ These sentences have been reformulated and more details given about the effects of rainfalls and temperature observed in the cited reference.

• Line 239: Sentence appears to contradict itself, can this be made clearer?

_ Sentence has been clarified. Here we meant that the French climate is similar from north to south regarding the Köppen-Geiger climate classification map.

• Lines 242-243: “Soil work” and “structuration”—see notes above.

_ See previous comments.

• Lines 253-260: I’m having trouble following the flow of the argument; please clarify. Also, on line 256, the statement is made that the study has a large geographic scale, whereas in line 239 the opposite is stated. Please clarify.

_ Paragraph has been detailed to clarify our reasoning. Concerning the geographic scale, we meant here that at the country scale of this work, environmental variables have more impact on communities’ structure than the crop rotations that are very similar across the sampled fields (majority of cereals). Line 239 on the contrary, we stated that France is not a large country compare to the worldwide scale studied in the cited reference (Nielsen et al. (2014)) to highlight that climatic variations in our work are less diverse than climatic variations at a worldwide scale.

• Line 266: “certain groups of years”. Specify the dates and/or date ranges.

_ See reviewer #1 comment.

• Line 271-272: “Their effects appeared to be positive or negative, probably depending on the PPN ecological niche.” This sentence is vague and requires considerable expansion; please discuss the specific positive and negative effects, and how these effects are or could be driven by a given ecological niche.

_ Sentence has been removed as discussion about soil physico-chemical variables impacts on PPN taxa occur in the following sentences.

• Line 273: “inputs”; please specify the inputs that you are referring to and how inputs are characterized to increase yield.

_ Sentence has been slightly modified. We meant here “fertilizers” that are used to enrich the soils for better plant growth.

• Line 274: “better quality” is subjective. What, specifically, constitutes quality, a change in quality, and what do these changes induce in nematode consortia?

_ “better quality” has been removed and we have detailed what we meant here. Positive effect of fertilization on PPN abundance may be due to more dense root systems that will allow a higher PPN development as the resource is abundant.

• Lines 285, 287: In the discussion crop rotations are described as having been removed from analyses, therefore it should not be possible to draw conclusions from them. Please clarify.

_ First sentence of the Conclusions section has been removed as it is now more detailed in the discussion about the crop rotations (see previous comments).

• Line 289: By “population” do you mean “abundance”?

_ Yes. The word “population” has been consequently modified by “abundance”.

• Lines 317-318, 320: It is safe to say that many readers will not be familiar with the national monitoring program. Within the regions listed, please specify field site locations, including the geolocations of the sites. In line 320, specify which regions, sites, and fields were used and note where changes occurred between years.

_ According to reviewer #1 comment, details about the French monitoring program for M. chitwoodi and M. fallax have been added.

It is not possible to give more details about the geolocation of the fields as this information is confidential for the French phytosanitary services and farmers agreed to participate to this work only on an anonymously basis.

As requested, informations were added to highlight where changes occurred between years (lines 366-367 of the track change version).

• Line 322: Specify the name of the agricultural data website, not just the reference. Were mean temperatures obtained for each field individually? If not, how were temperatures determined?

_ We specified the name of the website and gave more details about the data collections. Data were collected for each field individually from the closest meteorological station.

• Line 333: What is an “elementary soil core”? This is not terminology that I am familiar with.

_ In this work, it is a soil carrot over the first 30 centimeters of soil.

• Line 344: Should this be “sieves”? If so, please specify all sieve sizes as well as any standard protocols used.

_ More information has been added about the sieves and the whole extraction processes, following other comments from reviewer #1

• Line 345-346: “based on personal observations” is vague; specify the exact methodology or calculation required to determine the number of larvae per cyst.

_ In a previous work published by Garcia et al. 2018 in Nematology (DOI 10.1163/15685411-00003136) we investigated the number of juveniles and eggs in Heterodera cysts sampled in 7 fields in Burgundy during two consecutive years. A total of 33 cysts belonging to different species were individually crushed and the number of juveniles and eggs counted. The obtained mean juveniles + eggs per cyst was then used to convert cyst numbers of other French fields into juvenile and eggs numbers.

• Line 351: Please define “field surface”.

_ See previous comment.

Submitted filename: ResponseToReviewers.docx

Click here for additional data file.

6 Jan 2022

19 Feb 2022

Response to the points raised by the academic editor and reviewers

Dear editor,

Please find here a response to reviewer 3 and your comments and a revised manuscript according to all the points raised. All the comments have been addressed except the inclusion of metadata regarding PPN communities in fields where M. chitwoodi is present (see following comment) as we did not find such relevant information in the scientific literature. However, we have slightly modified the text to consider those comments.

Sincerely

Dr. Nathan Garcia

Editor comments:

• I agree with Reviewer 3 that the inclusion of supporting metadata from fields that are indeed infested with M. chitwoodi could potentially strengthen the discussion and value of your results. I acknowledge that your study was initially designed to identify parameters that could aid the development of a focused M. chitwoodi monitoring scheme, but as you (fortunately for the owners of the sampled fields!) did not detect the species, you could consider putting less emphasis on this objective. As we are far from understanding which parameters/variables are key to the distribution of nematode taxa in general, the results on the plant parasitic nematode community is valuable on its own, even though you did not find M. chitwoodi.

_ We agree that metadata about nematode communities in fields infested with M. chitwoodi would be an interesting implementation in this study. However, we did not find any relevant publication in the literature to do so since we only found publications focusing on experimental design instead of agricultural fields. That appear not suitable for comparison with our study since the experiments in those studies had a major impact on the nematode communities and M. chitwoodi abundance prior to any other parameter. Furthermore as M. chitwoodi is regulated in many countries, fields that are known to be infested are usually under specific management that depend on the countries legislation. These managements implies that the whole nematode communities are drastically impacted as there is no specific management targeting M. chitwoodi alone. Thus, to our opinion, even those data would not be very relevant to compare with our work because they would not displayed the nematode communities commonly found in agricultural soil as we are doing in this study.

However, we have followed your suggestion to put less emphasis on M. chitwoodi in the revised version of the manuscript as we totally agree that impact of the studied variables on PPN communities is valuable on its own. We still believe that this kind of study can help the phytosanitary services to target the fields in their monitoring scheme as we know that PPN communities have an impact on M. chitwoodi abundance (since we observed it in Garcia et al. 2018 (DOI: 10.1111/ppa.12914)). But as you mentioned, we are far from understanding the whole mechanisms of soil nematode communities distribution. Thus several sentences of the abstract and introduction have been removed or modified.

• L. 29: ”anthropic … variables” sounds a bit odd; please rephrase to “cultivation practices and environmental variables”.

_ Agree, the sentence has been modified and according to the following comments, “anthropic” has been replace by the appropriate term for the whole manuscript.

• Throughout manuscript: Do not use “anthropic”, please change to “anthropogenic” or, if appropriate, use “cultivation practice”, which more accurately describes the nature of anthropogenic activities.

_ See previous comment

• L. 35: Again, there is no such thing as “anthropic soil variables”.

_ See previous comment. Here we meant “the cultivation practices that have an impact on soil” (such as tillage).

• L. 44: In soil, I would claim that nematodes belong to the microfauna (as defined as metazoan with a body width ≤100 µm). If you classify according to body length, nematodes belong to the mesofauna. However, the body width is much more relevant, as it determines which soil pore opening are accessible to the nematodes.

_ We agree but both reviewers 1 and 2 considered that nematodes belong to mesofauna. We have remove this terms from the sentence.

• L. 47: Like reviewer 1, I am also puzzled by the “natural abundances”; what would define “unnatural abundances” then? Better to state that most PPNs are not damaging to crops at the abundances commonly found in French agricultural soils (or something equivalent).

_ Agree, this is exactly what we meant here and the sentence has been modified accordingly.

• Table 1: Provide more information in the table legend; e.g. that data depict the mean of 35 and 31 fields surveyed in 2015 and 2016, respectively, and clarify what “Prevalence” and “W” refer to.

_ Table legend has been detailed accordingly.

• Fig. 1: In l. 123-125, you mention that Fig. 1 includes the cultural practices, but I only see environmental variables, no reference to cultivation practices. Please clarify.

_ Here we meant that, regardless the outcome of the factorial analysis (i.e. the factorial map presented in Fig.1), cultural practices were implemented to run the analysis and that for fig. 1, only the cultural practices of the sampling year were implemented (not those of the past years that are implemented to run the analyses shown in S1Fig.). As tb-RDA allow a selection step thanks to a permutation test, Fig.1 display only the significant variables which are, as you mention, only environmental variables.

The text has been modified in order to clarify.

• Table 2: Please explain the variables (C/N, Herbi, Zn) in the legend. The meaning of “Year period” should also be explained. Also describe which type of model the table depict; in essence, the table and figures should be self-explanatory.

_ Legend has been substantially detailed and the model type (i.e. Poisson GLMM) has been added in title.

• L. 210-211: Which better host plant?

_ It is impossible to say as species of this family feeds on a very large host range, including cereals that have mainly been grown during 2016 for the sampled fields, but also many weeds that have not been considered in this study. However, we have slightly detailed what we meant in the sentence.

• L. 215-236: You do not discuss your own results here; please do so.

_ Agree. The whole paragraph has been detailed to link the literature knowledge to our own results.

• L. 216-226: Are the referenced studies on heavy metal effects conducted in soil or in other matrices/media? Generally, the bioavailability and negative effects of heavy metals on soil organisms are dramatically reduced in soil compared to e.g. liquid media, so this information is quite important to evaluate the comparability to your study.

_ References 16, 33, 34 studied heavy metal effect on nematode communities in soil and references 31 and 32 studied their effect on C. elegans on a growing media but observed detrimental effect even at low concentration. In our discussion, references 16, 33 and 34 are cited to be compared to our results and while reference 31 and 32 are cited to explain the physiological mechanisms involved. As the paragraph has been modified accordingly to the previous comment, we also included these elements.

• L. 218: Please add a reference that shows negative correlation between Zn conc. and PPN abundance.

_ L.218, we were discussing about our own results. We agree that it was not clear and thus the sentence has been modified.

• L. 245: Coming from a small country, I find that France is a rather large country. In any case, the important issue here is not the size of the country, but climatic variation, so I suggest you omit reference to the size of the country and focus on climatic variation across the sampled regions.

_ Agree, and this is what we meant. Reference to the country size has been removed in the text.

• L. 249: “field surface area”.

_ Sentence has been replaced

• L.250-51: “… we did not sample more samples in the larger fields), but sampled seven soil core over the longest diagonal in all fields”

_ Sentence has been replaced

• L. 252: “surface area affected PPN communities”

_ Sentence has been replaced

• L. 269: “contrasts”

_ Corrected

• L. 368: “surface area”

_ Sentence has been modified

• L. 388: Please define “MCA”

_ MCA (for multiple correspondence analysis) has been defined

• L. 402 and 403:”surface area”

_ Sentences has been modified

• L. 405: Define “SW” and “CI”

_ SW (for sum of weight) and CI (for confident interval) have been defined.

Reviewer #3 comments:

• In the abstract, introduction and discussion the authors emphasize that the obtained knowledge described in this work can facilitate management of the establishment of an introduced species (M. chitwoodi). Basically the aim is to look for parameters to develop an more focussed sampling scheme to monitor M. chitwoodi/M. fallax infestations. Unfortunately in none of the sampled fields M. chitwoodi was detected. It would have been an added value if M. chitwoodi-infested fields were included in this study. If metadata are available for known infested fields it would be useful to compare these with the results from this study and add this comparison to the discussion. With the provided data no conclusion can be made regarding higher possibilities for establishment of M. chitwoodi.

_ See first comment from editor.

• Statement in line 49-51 is not backed by references given, other refs should be used

_ References used here has been modified.

Submitted filename: ResponseToReviewers.docx

Click here for additional data file.

23 Feb 2022

Diversity of plant parasitic nematodes characterized from fields of the French national monitoring programme for the Columbia root-knot nematode

PONE-D-21-19128R2

Dear Dr. Garcia,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Mette Vestergård, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reading through the manuscript I noticed some errors listed below that you should amend when you do the final review of the manuscript:

L. 111: ”These”

L. 191: ”the presence of the same PPN…”

L. 251: “cores”

L. 301: There is no such thing as a “”PPN ecosystem”; please rephrase – I presume you mean “PPN communities”

Reviewers' comments:

28 Feb 2022

PONE-D-21-19128R2

Diversity of plant parasitic nematodes characterized from fields of the French national monitoring programme for the Columbia root-knot nematode

Dear Dr. Garcia:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Kind regards,

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on behalf of

Dr. Mette Vestergård

Academic Editor

PLOS ONE