Soil Nematode Trophic Group Composition and Influence on Growth of Amaranthus palmeri and Parthenium hysterophorus

Weeds have a significant impact on agricultural systems. They not only cause a loss in crop yield by competing with them for resources, but they can also serve hosts for several pests and parasties such as plant parasitic nematodes casusing additional crop loss. The aim of this study was to analyze plant-nematode feedback in two major weeds, Amaranthus palmeri S Watson and Parthenium hysterophorus L. First, a field survey was conducted to determine the rhizosphere nematode trophic groups associated with these two plants in the summer of 2020 and 2021. Then a 6-week greenhouse study was conducted where the two weed species were treated with nematode communities extracted from their respective rhizospheres. Results from this study show that both weeds harbored a high number of herbivore nematodes, followed by fungivore and bacterivore nematodes. Total number of these nematodes were highly influenced by total sol carbon, pH and salinity. Under greenhouse conditions, the nematode treatment did not have any impact on the growth of P. hysterophorus but A. palmeri plants treated with nematodes had significantly higher above ground biomass. In conclusion, plant-nematode relationships are complex. Given the extent of direct damage caused by these weeds and plant parasitic nematodes in global crop production, the weeds-nematode feedback warrants further detailed studies.

in the presence of other free living soil nematodes are known to improve plant growth by stimulating microbial activity and consequently increasing N mineralization in soil benefitting the plants (Gebremikael, Steel, Buchan, Bert, & De Neve, 2016). Most of the past studies on weed-nematode interactions have mostly focused on the few numbers of economically important plant parasitic nematodes, while the interaction between weeds and free-living soil nematodes, which occupy key positions in the soil food-web (bacterivores, fungivores, omnivores, and predators), are not clear.
In this study, the impact of two major weeds, Amaranthus palmeri S Watson (Palmer amaranth) and Parthenium hysterophorus L. (Parthenium, whitetop weed, false ragweed) on the soil nematode community and the influence of soil nematodes on the growth of these two plant species was analyzed. Both A. palmeri and P. hysterophorus are the most aggressive and herbicide-resistant weeds worldwide (Heap & Duke, 2018;Kistner & Hatfield, 2018). They invade agriculture fields, pastures, roadsides, and natural systems (Adkins & Shabbir, 2014) and cause a significant reduction in crop growth and yield. These weeds are also known to be hosts of root-knot nematodes (Meloidogyne spp.) (Chitambo, Haukeland, Fiaboe, & Grundler, 2019;Datta, 2006), which cause a significant decline in crop growth and yield, especially vegetables, and cause serious economic losses (Collange, Navarrete, Peyre, Mateille, & Tchamitchian, 2011). This research builds on the work by (Lopez et al., 2021) who reported high number of plant parasitic nematodes on P. hysterophorus and other coexisting weeds in south Texas. In this study, we specifically aimed to answer two questions: 1) How do the two weeds influence the trophic groups of soil nematodes in their rhizosphere? 2) How do the soil nematode trophic groups influence the growth of these two species? To answer these questions, we conducted this study in two phases: 1) field survey of roots and analysis of the rhizosphere soil samples under these two weeds and 2) a greenhouse growth study of the two weed species treated with the soil nematode communities collected from their respective rhizosphere in the field.

Field Study
The field surveys in this study were conducted in 5 different organic vegetable farms in the Lower Rio Grande Valley (LRGV) in south Texas during the summer (June-July) of 2020 and 2021. The LRGV has a semiarid sub-tropical climate with mild winters and hot summers. Vegetable growing season in this region spans from September through May and fields are left fallow (weedy or kept bare with continuous cultivation for weed removal) during summer (June-August).
At each farm we randomly selected 25 plants of each plant species to assess nematode damage symptoms in roots as well as the leaves. We analyzed the plants for aboveground symptoms such as leaf color and plant size and belowground symptoms such as stunted root growth, galls, lesions, and root rotting. We then collected approximately 100 g of rhizosphere soil from five different locations with dense growth A. palmeri and P. hysterophorus at 0-15 cm depth. The soil samples were placed in plastic bags and transported to the lab in a cooler for further analysis. In the lab, 50 g of soil were stored in the refrigerator until further analysis of soil edaphic properties and 50 g of soil were used to extract and analyze the soil nematode community.

Nematode Extraction and Analysis
The sucrose-centrifuge method (Jenkins, 1964) was used to extract the nematodes from the soils. To extract nematodes, 50 g of soil was mixed with DI (deionized) water in a beaker and was sieved by placing 106 µm and 38 µm sieves on top of one another. The sediments retained in 38 µm micrometer sieve were poured into a 50 ml centrifuge tube which were centrifuged at 3500 rpm for one minute. The supernatant was then decanted. Sucrose solution was added to the pellet in the 50 ml tube and mixed. The samples were then centrifuged at 3500 rpm for one minute to separate the nematodes from the soil. The supernatant with nematodes was poured into a 32 µm mesh and washed to clean the sucrose from the nematodes. The extracted nematodes were stored overnight in a 4 ºC refrigerator. The nematodes were then viewed under an inverted microscope (Leica DMi1, Buffalo Grove, IL) and counted. The nematodes were identified morphologically and grouped based on their trophic groups: 1) plant-parasitic/herbivores, 2) bacterivores, 3) fungivores, and 4) predators/omnivores.

Soil Analysis
The soil samples were analyzed for salinity, organic matter (OM%), pH, and total carbon (C) and nitrogen (N). Soil moisture was measured using the gravimetric method. Organic matter was determined by the dry combustion method (500 o C for 4 hours). Soil pH was measured with a benchtop pH meter (OAKTON ION 700 Thermo fisher scientific, Waltman, MA, USA) in 1:2 (soil:DI water solution). Salinity was measured with an Accumet Conductivity Meter (AB200 Thermo fisher scientific, Waltman, MA, USA) in 1:2 (soil:DI water solution). Total C and N was measured with a C/N analyzer (928 Series Macro Determinator, LECO, St. Joseph, MI, USA).

Greenhouse Study
In July 2021, a 6-week greenhouse study was conducted to determine the impact of nematodes on the two weed species, A. palmeri and P. hysterophorus. A total of 24, one week old seedlings of both weed species (12 each) were randomly selected and transplanted in 1 liter plastic pots filled with soil collected from local farms and sterilized in an autoclave and mixed with perlite (5:1 volume). The soil had 1.32% organic matter, 1.24% total carbon and 0.097% total nitrogen. Six pots of each weed species were treated weekly with nematode communities extracted from the rhizosphere of each weed species in the field. The nematode composition and number of nematodes added to the nematode treatment pots are presented in Table 1. Stomatal conductance and chlorophyll fluorescence were measured weekly using the LI-600 Porometer/ Fluorometer (LI-COR Biosciences, Lincoln NE, USA). After 6 weeks, when the A. palmeri plants started producing flowers, they were destructively harvested and dried in an oven to constant weight (70 o C, 74 hours) to determine the total shoot biomass and biomass allocation pattern in the two weed species. To determine the nematode host status of the two weeds, 25 g of roots were randomly collected from each of the nematode treatment plants and used to extract root nematodes following the root incubation method in (Hallman & Viaene, 2013). The roots were rinsed with water, cut into small pieces, and sliced by cutting cross sectionally. Roots were then placed (fully submerged) in beakers with 100 ml DI water at 24 o C for two weeks. During the two-week period, 1 ml water was pipetted from each beaker and observed under the inverted microscope every 3 days. After 2 weeks, the roots were discarded. Since we did not find any nematodes in any of the root samples during the 2 weeks period, no further analysis was done. We also analyzed the soil samples in the pots at the end of the 6-week period to determine the impacts of the weed-nematode associations on the abundance of nematodes in each trophic groups and plant growth. 50 g of soil samples was collected from each treatment pot and the different types of nematodes were extracted and enumerated as outlined above.

Data Analysis
For the soil nematode community analysis, relative abundance of each trophic group, Shannon diversity index, evenness index, Simpson's index, and nematode channel ratio were calculated as shown in Table A1. Differences in the soil properties between the two years were compared with ANOVA. The relative abundance of different nematode groups was not different between the two years, except for predator nematodes in A. palmeri, so the data for the two years were combined and analyzed together. Data on soil nematodes were log transformed (log x + 1). We used forwards stepwise model selection using step AIC to find the best model with which to describe the current dataset. Multiple linear regression analysies (backward elimaination) was performed to evaluate the relative importance and effects of soil variables on the soil nematode trophic groups and community indices. The Akaike information criterion (AIC) was used to identify variables for model selection. Soil variables that were selected by the final RDA models were used to build models.
For the greenhouse study data, canopy cover, plant height, and stomatal conductance were not normally distributed, and thus were log transformed. Student t-test was done to compare the plant measurements in the jas.ccsenet.org Journal of Agricultural Science Vol. 14, No. 11;2022 nematode and control treatments for each species. Results were considered significant if P ≤ 0.05. All data was analyzed using JMP Pro statistical software.

Field Survey of A. palmeri and P. hysterophorus
There were some differences in the soil characteristics in the rhizosphere of the two weeds (Table 2). Soil pH was slightly alkaline. While there was no significant difference unde the two weeds in 2020 it was significantly higher under A. palmeri in 2021 (P = 0.015). Soil salinity was significantly higher under A. palmeri in 2020 (P = 0.001).
There was no significant difference in other soil properties between the two weeds in both the years. A total of 57,910 nematodes of 22 different morphospecies were recorded in the rhizosphere soil samples of the two weeds species. Of which, there were 9 herbivore, 5 fungivore, 3 bacterivore, 1 predator, and 4 unidentified nematode species (features not possible to identify, probably due to damage during extraction). While there was a difference in the relative abundance of the different nematodes in the rhizosphere of the two weed species, herbivores were the most dominant group of nematodes in both the years (Table 3). There was no significant difference in the different nematode types under the two weed species except for the predator nematodes under A. plameri in 2020. In 2020, A plameri had significantly higher number of predators (P = 0.018) and lower number of herbivores, though not statistically significant. Among the different herbivores, Helicotylenchus spp., Tylenchus spp., and an unknown species of herbivore nematodes were the dominant ones in both weed species (Table 4). Pratylenchyus spp., Paratylenchus spp., and Mesocriconema spp. were also found in the soil samples, but their numbers were relatively low.

Influence of Soil Parametres on Nematode Communities
Soil neamtodes are highly influenced by soil biotic and abiotic conditions. They are known to have a narrow range of favorable soi conditions such as moisture, which are highly influenced by soil particle size (Simons, 1973). In this study, soil variables such as total carbon, pH, salinity, organic matter, and total nitrogen in soil had different impact to the different nematode trophic groups and their community indices.
Results from this study show that soil carbon has a significant influence on the soil nematode communities. Increase in soil carbon content resulted in higher numbers of nematodes. Similarly, total number of herbivore and fungivore nematodes also increased linearly with the increase in soil carbon, while we did not see a significant association with the bacterivore nematodes. Bacterivore nematodes declined with the increase in soil pH. Soil pH between 5-7 are reported to be favorable for soil nematodes (Matute, 2013;Warner, 2009). In this study, the average soil pH in the farms was 7.54, this slightly alkaline pH could have reduced bacterivore nematodes. Similarly, bacterivore nematodes also increased linearly with soil salinity. The positive relationship between bacterivores and soil salinity was an unexpected result showing the bacterivore nematodes in this study are tolerant to soil salinity under field conditions. Soil nitrogen enrichment is reported to increase the abundance of of bacterivores and suppressed fungivores and predators (Song et al., 2016), in this study we did not find a strong influence of nitrogen in any of the nematode trophic groups. This could be the result of heavier soil texture or inherently low nitrogen content in these study sites.
Though not very strong, soil variables also had an influenc on the nematode community indices. Shannon diversity index increased the soil carbon content and salinity. Evenness had quadratic relationship with soil nitrogen. NCR value which indicates the organic matter decomposition pathway was around 0.5 which shows that the summer soil conditions (typically hot and dry) are not ideal for the bacterial community. The positive linear relationship between soil carbon and NCR indicates improving soil carbon content can promote the bacterial community. Overall, these resuls show that soil carbon is the key indicator of soil nematode community composition in organic vegetable systems.

Influence of Nematodes on the Growth of the Two Weeds
Results from the greenhouse study show that herbivore nematodes do not necessarily lead to decline in plant health. Nematode species identity is more critical to plants as suggested by (Brinkman, Duyts, & Van der Putten, 2005b). These results are similar to previous studies reporting no difference in the shoot biomass with nematode treatment (Brinkman, Duyts, & van der Putten, 2005a;Brinkman et al., 2005b). A. palmeri plants treated with nematodes had higher shoot biomass. This could be the result of increased number of bacterivores, fungivore, and predator nematodes which aid in nutrient cycling (Ferris, Venette, & Scow, 2004). The total number of plant parasitic nematodes declined in both weed species while the beneficial nematodes bacterivores and fungivores increased in number in the pot experiments at the end of the study period. Lower number of herbivores in soil along with the absence of nematodes in the roots indicate that the two weed species do not serve as direct hosts of the plant parasitic nematodes recorded in this study. A possible explanation for the high number of plant parasitic nematodes in the rhizosphere of the two weed species in the field could be the presence of other smaller weeds in the understory of these two weed species. The other possibility is the release of volatiles by these two weed species which attract plant parasitic nematodes (Reynolds et al., 2011), but this weeds-volatile-nematode interaction in A. palmeri and P. hysterophorus requires further research.
In conclusion, results form this study show that the two weeds A. palmeri and P. hysterophorus harbor a high number of herbivore nematodes to whom they do not directly serve as hosts. In addition, abundance of soil nematodes was highly dependent on the soil conditions, particularly soil carbon and pH. A. palmeri, which is described as "one of the most widespread, troublesome, and economically damaging agronomic weeds in the southeastern U.S." (Ward et al., 2013), can potentially attract high number of plant parasitic nematodes. Thus, identifying nematode species is crucial in adopting a both weed and nematode management technique in agricultural fields. With the increased research interest in manipulating the plant microbiomes as an invasive plant/weed management tool (Shahrtash & Brown, 2021), more research is needed on the plant-soil feed back in both A. palmeri and P. hysterophorus and their role in shaping the soil nematode community. In addition, the morphological identification of the nematodes used in this study does not provide full information on the nematode species, thus there is a need for further research in weed mediated plant-soil feedback using molecular techniques for proper identification of plant parasitic nematodes and effective management of both weeds and nematodes in agricultural systems.