Reaction of Stylosanthes spp. 'Campo Grande' to Pratylenchus brachyurus and Meloidogyne javanica and analysis of the histopathology and histochemistry of their interactions

In an integrated nematode management system, the best results are generally obtained by rotating crops with resistant plants, antagonists, or bad hosts of parasites. Some reports indicate that Stylosanthes spp. have the potential to control nematodes. Thus, this study aimed to assess the reaction of Stylosanthes spp. 'Campo Grande' to Pratylenchus brachyurus and Meloidogyne javanica penetration and reproduction at different inoculum levels and examine the histopathology and histochemistry of parasitized plants. Stylosanthes did not prevent P. brachyurus penetration in roots. However, the number of penetrated nematodes was lower than that in soybean from 17 days after inoculation onwards. The numbers of second-stage and third-/fourth-stage juveniles of M. javanica in Stylosanthes roots were close to zero, and no females were observed. Assays conducted using increasing levels of inoculum (P. brachyurus or M. javanica) showed that Stylosanthes was resistant to parasites, with a maximum reproduction factor of 0.59 for P. brachyurus and 0.07 for M. javanica. Histopathological analysis showed the presence of P. brachyurus in Stylosanthes, but without rupture of plant cells. M. javanica individuals were not observed. Histochemistry revealed the presence of phenolic compounds in the epidermis of Stylosanthes and proteins in plant cells. These results show that Stylosanthes spp. 'Campo Grande' can be used in crop rotation programs in �elds with mixed infestation of P. brachyurus and M. javanica.


Introduction
Nematodes are among the most important plant pathogens, given their economic impact, di cult management, and growing presence in agricultural elds.Plant-parasitic nematodes affect large extensions of cultivated lands, including soybean crops, a major agricultural commodity worldwide.
P. brachyurus is one of the most common nematode species in tropical countries.In Brazil, yield losses of up to 21% have been reported in infested crops (Franchini et al. 2014).Nematodes of the genus Pratylenchus are migratory endoparasites that cause root lesions as infectious forms of the parasite feed and move within roots (Elhady et al. 2017).
Meloidogyne is considered the most important genus of plant-parasitic nematodes on a global scale because of its wide distribution, broad host range, and completion of several life cycles in host plants (Nyczepir and Meyer 2010).In Brazil, the most important species parasitizing soybean are M. javanica and M. incognita, which can cause losses of up to 55% under severe infestation conditions (Silva et al. 2020).Females of this nematode genus are sedentary endoparasites that establish complex feeding sites in host roots, promoting cell division and increasing the size of cortex cells, thereby resulting in the formation of galls (Elhady et al. 2017).The effects of such lesions are re ected in aerial parts through stunted growth, leaf chlorosis, and plant death.
Soybean cultivation has expanded to the Brazilian Cerrado, where soybean/maize rotation systems are widely used.However, both crops can raise population levels of P. brachyurus and M. javanica (Tavares-Silva et al. 2017; Zavislak and Araújo 2018), necessitating strategies to control these nematodes.The use of antagonist plants in rotation with or succession to soybean is one of the most recommended techniques for nematode management.Antagonist plants produce substances capable of repelling or inhibiting other organisms (Chitwood 2002).Non-hosts and bad hosts can also be valuable in rotation systems (Ferraz and Brown 2016).
Legume plants are widely studied for their ability to control nematodes, x atmospheric nitrogen, and enrich the soil with organic matter (Ferraz et al. 2012).Although they can provide numerous advantages to cropping systems, some legume species are not well adapted to low-fertility sandy soils, which are common in the Brazilian Cerrado and sandstone regions.In Mato Grosso State, where 25% of soils are sandy, P. brachyurus has been detected in 96% of soybean elds (Ribeiro et al. 2010;Kappes and Zancanaro 2014).This scenario underscores the need to identify legume species that are better adapted to such conditions.
The forage legume Stylosanthes exhibits interesting characteristics for use in soybean/maize systems.With a deep root system, Stylosanthes has the ability to grow in poor sandy soils, promoting the cycling of minerals from deeper soil layers and increasing soil organic matter concentrations (Schultze-Kraft et al. 2018).The species has also been shown to e ciently reduce the levels of different nematodes, such as P. brachyurus, M. javanica, and R. reniformis (Gardiano et

General experimental information
In all experiments, experimental units consisted of polystyrene cups containing 500 cm 3 of a 1:1 (v/v) mixture of soil and sand previously autoclaved for 2 h at 120°C.The P. brachyurus inoculum was obtained from a pure population maintained on soybean 'M6410 IPRO' and extracted according to Coolen and D'Herde (1972).The M. javanica inoculum was obtained from a pure population kept on tomato 'Santa Clara' and extracted according to Hussey and Barker (1973) with the modi cations proposed by Bonetti and Ferraz (1981).After extraction, nematodes were counted in a nematode counting chamber (Peters' chamber) under a light microscope at 40× magni cation.Experiment 1: Penetration of P. brachyurus and M. javanica in roots of Stylosanthes spp.'Campo Grande' The experiment was conducted in March 2018 in a greenhouse (23°47′28.4″S53°15′24.0″W,379 m a.s.l.).Mean minimum, average, and maximum temperatures were 21.24, 25.38, and 30.48°C, respectively.A completely randomized design with a 2 × 4 factorial arrangement was used.The rst factor was plant species (Stylosanthes and soybean), and the second factor was evaluation period (7,12,17, and 22 days after inoculation, DAI), with ve replications per treatment.
Firstly, seeds of Stylosanthes spp.'Campo Grande' were planted in polystyrene trays containing commercial substrate (Bioplant®).Fifteen days after germination, seedlings were transplanted to other pots, and seeds of soybean 'M6410 IPRO' were sown in the same experimental units.After ve days, pots were inoculated with 2 mL of a suspension containing 500 individuals of P. brachyurus or 2000 eggs + second-stage juveniles (J2) of M. javanica.The inoculum was deposited directly onto plant roots, which were subsequently covered with soil.At 7, 12, 17, and 22 DAI, plants were evaluated for nematode penetration in roots.For this, plants were carefully removed from pots.Then, the root system was collected, washed in water, placed on absorbent paper to remove excess water, and weighed on a semi-analytical scale to determine the fresh weight.Subsequently, roots were stained with acid fuchsin (Byrd Júnior et al. 1983).After staining, temporary slides were prepared with root fragments, using the whole root.Slides were examined under a light microscope at 40× magni cation for quanti cation of penetrating nematodes.For samples inoculated with M. javanica, in addition to total nematode number, we determined the numbers of J2, third-and fourth-stage juveniles (J3/J4), and females.Experiment 2: Effect of increasing inoculum levels of P. brachyurus and M. javanica on Stylosanthes spp.
The experiment was set up as described above for Experiment 1. Stylosanthes spp.'Campo Grande' plants were inoculated with different initial levels as mentioned above.Inoculum viability was tested by inoculating soybean with 500 individuals of P. brachyurus or 2000 eggs + J2 of M. javanica.
At 80 DAI of P. brachyurus and 60 DAI of M. javanica, plants were removed from pots and evaluated for root fresh weight and nematode reproduction.Roots were subjected to the nematode extraction processes described above, and nematodes were counted in a Peters' chamber under a light microscope.The nal nematode population was divided by the root fresh weight to obtain the population density (number of nematodes per gram of root).Then, the reproduction factor (RF) was calculated according to the equation proposed by Oostenbrink (1966), as follows: RF = Final population/Initial population.

Experiment 3: Histopathological and histochemical analyses
For visualization of the histopathology underlying the interaction of Stylosanthes spp.'Campo Grande' with P. brachyurus and M. javanica, a third experiment was conducted in a greenhouse (23°25′38″S 51°56′15″W, 551 m a.s.l.) in October 2021 (minimum, average, and maximum temperatures of 17.80, 22.39, and 27.06°C).The design was completely randomized with four treatments (Stylosanthes + P. brachyurus, Stylosanthes + M. javanica, soybean + P. brachyurus, and soybean + M. javanica) and ve replications per treatment.The experiment was set up as described above for Experiment 1. Plants were inoculated with 500 individuals of P. brachyurus or 2000 eggs + J2 of M. javanica.At 30 DAI, plants were carefully removed from the pots, the aerial part was discarded, and the roots were washed and used for histopathological analysis.
Samples were prepared according to the method for preparation of histopathological sections described by Pergard et al. (2005).Twenty root segments per replication were cut into 0.3 mm thick slices, placed in 1.5 mL microtubes, and immersed in 1 mL of xative (2% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.00) under continuous agitation.Tissues were then dehydrated in an increasing ethanol series and embedded in resin (Technovit ® 7100), following the manufacturer's protocol.Subsequently, samples were cut into 5-7 µm thick sections using an ultramicrotome (Lupetec ® MRP 2015).Sections were xed on microscope slides by using drops of distilled water and drying on a heating plate.Samples were stained with 0.5% toluidine blue for histological analysis.For histochemical analysis, 10% ferric chloride was used to detect phenolic compounds (Johansen 1940) and xylidine Ponceau to detect proteins (Cortelazzo and Vidal 1991).Photographs of root sections were taken using a digital camera (Moticam 1080) attached to an optical microscope (Motic BA310E).

Statistical analysis
Data from penetration experiments and initial inoculum levels were analysed for normality by the Shapiro-Wilk test.Nematode numbers were transformed to .Then, the data were subjected to analysis of variance at the 5% signi cance level.Plant species were compared using Bonferroni's t-test (p < 0.05).Differences between evaluation times and inoculum levels were analysed by Tukey's test, both at p < 0.05.Statistical analyses were performed using Sisvar software (Ferreira 2014).

Results
Experiment 1: Penetration of P. brachyurus and M. javanica in roots of Stylosanthes spp.'Campo Grande' Plant species × Evaluation period effects on total P. brachyurus number were signi cant (Table S1, Supplementary material).Total nematode number was higher in soybean than in Stylosanthes spp.'Campo Grande' at 17 and 22 DAI (Table 1).Furthermore, in Stylosanthes spp.'Campo Grande', total nematode number did not vary with time, whereas, in soybean, nematode numbers increased over time, being higher at 22 DAI than at 7 and 12 DAI (Table 1).DAI, days after inoculation, CV, coe cient of variation.
For M. javanica, the interaction effects of factors on J2 number were signi cant (Table S2, Supplementary material).J2 number was higher in soybean than in Stylosanthes spp.'Campo Grande' at all evaluation periods, except at 7 DAI, when there were no differences between plant species (Table 2).In Stylosanthes spp.'Campo Grande' roots, J2 number did not vary over time, with means close to zero.In soybean roots, J2 number was highest at 17 DAI (Table 2).At 17 and 22 DAI, J3/J4 were detected in soybean roots only, and their numbers were higher at 22 DAI (28.4) than at 17 DAI (11.2) (data not shown).Furthermore, females were identi ed in soybean roots at 22 DAI (data not shown).By contrast, no females were observed in Stylosanthes spp.'Campo Grande' roots.An increase in P. brachyurus inoculation levels in uenced the RF (Table 3).The RF was higher in treatments with initial inoculum levels of 250 than in those with initial levels of 1000 in both trials.The RF of P. brachyurus on Stylosanthes spp.'Campo Grande' was lower than one under all conditions (Table 3), whereas the RF on soybean was 2.0 in Trial 1 and 10.95 in Trial 2, con rming the viability of the inoculum (data not shown).For M. javanica, there was no signi cant in uence of initial inoculum level on the study variables (Table 4).The RF on soybean was 4.58 in Trial 1 and 8.32 in Trial 2 (data not shown), whereas that on Stylosanthes spp.'Campo Grande' ranged from 0 to 0.07 (Trials 1 and 2).This nding shows that the plant was resistant to the root-knot nematode.

Experiment 3: Histopathological and histochemical analyses
The histopathological study con rmed the presence of M. javanica in the roots of Stylosanthes spp.'Campo Grande' at 30 DAI (Fig. 1A).In soybean, fully developed females of M. javanica were identi ed, together with feeding sites composed of hypertrophied, multinucleated cells with thick walls, dense cytoplasm, and small vacuoles (Fig. 1B).The presence of P. brachyurus in Stylosanthes spp.'Campo Grande' (Fig. 1C) and soybean (Fig. 1D) at 30 DAI was also con rmed.However, ruptured cortex cells, resulting from intracellular movement of nematodes, were only observed in soybean.
We did not identify phenolic compounds in soybean cells parasitized by M. javanica (Fig. 2A) or P. brachyurus.Although epidermal cells were not penetrated by nematodes, a phenolic-rich layer was formed under the epidermis of Stylosanthes spp.'Campo Grande' (Fig. 2B).
Protein accumulation in cells was identi ed in Stylosanthes spp.'Campo Grande' roots (Fig. 3A and B) but without the formation of feeding sites by M. javanica or cell damage by P. brachyurus.By contrast, in soybean roots, M. javanica developed completely, and root cells lacked proteins (Fig. 3C).

Discussion
Stylosanthes spp.'Campo Grande' was not a host to P. brachyurus, in agreement with the ndings of previous studies, which observed RF values lower than one for P. brachyurus (Santos et al. 2011).The RF of Pratylenchus zeae on Stylosanthes spp.'Campo Grande' was 0.0 and 0.1 after 90 and 120 days of cultivation, respectively (Obici et  The ndings showed that Stylosanthes spp.'Campo Grande' acts as a bad host to M. javanica.Nematodes were poorly attracted to the plant, and, after penetration, J3/J4 development was delayed and females were not formed (Table 2, Fig. 2A).Thus, nematodes were not able to complete their life cycle.Stylosanthes spp.'Campo Grande' showed an RF value lower than one, even with increasing levels of initial inoculum (Table 4).
A previous study assessed the e cacy of Stylosanthes capitata in the control of M. javanica.The plant reduced total nematode number and population density by 99% and 87%, respectively, compared with soybean (Miamoto et al. 2016).In the referred study, the authors found that the plant had the lowest RF among other legumes with antagonistic potential, such as Crotalaria spectabilis.Other studies have also reported on the potential of Stylosanthes spp.for M. javanica management (Charchar et al. 2009;Sharma 1984).
The plant defence mechanism against nematodes may result in delay or non-formation of feeding sites (Machado et al. 2012).This is achieved by changes in metabolite concentrations in roots, such as by an increase in phenolic compound synthesis, as observed in the epidermis of Stylosanthes 'Campo Grande' (Fig. 3B).Phenolic compounds comprise a variety of polyphenols, such as simple phenols, phenylpropanoids, avonoids, tannins, and quinones.These results show that Stylosanthes spp.'Campo Grande' can be used in crop rotation systems in elds infested with P. brachyurus and/or M. javanica, behaving as a bad host or non-host to nematodes.

(
A) Absence of Meloidogyne javanica (Mj) in roots of Stylosanthes spp.'Campo Grande' at 30 days after inoculation (DAI).(B) Presence of Mj and formation of giant cells (GC) in soybean roots at 30 DAI. (C) Presence of Pratylenchus brachyurus (Pb) in roots of Stylosanthesspp.'Campo Grande' at 30 DAI. (D) Presence of Pb in soybean roots at 30 DAI; the red arrow indicates ruptured cortex cells resulting from intracellular movement of nematodes.

Figure 2 (
Figure 2 al. 2012; Obici et al. 2011; Rodrigues et al. 2014; Miamoto et al. 2016; Vedoveto et al. 2013).It should be noted, however, that the mode of action of the plant against nematodes has yet to be elucidated.

Table 3
Total number, population density, and reproduction factor of Pratylenchus brachyurus on Stylosanthes spp.'Campo Grande' and plant root weight at 80 days after inoculation with increasing nematode levels.
Note: Means within a column followed by different lowercase letters differ signi cantly by Tukey's test (p < 0.05).Original means were transformed to for statistical analysis.The absence of letters indicates no signi cant differences among treatments.TN, total nematodes; PD, population density; RW, root weight; RF, reproduction factor; CV, coe cient of variation.

Table 4
Total number, population density, and reproduction factor of Meloidogyne javanica on Stylosanthes spp.'Campo Grande' and plant root weight after 60 days of inoculation with increasing nematode levels.
Note: The absence of letters indicates no signi cant differences among treatments.TN, total nematodes; PD, population density; RW, root weight; RF, reproduction factor; CV, coe cient of variation.
(Rodrigues et al. 2014ved that P. brachyurus population density decreased by 67.6% and 76.5% in the roots of soybean grown in succession to Stylosanthes spp.'CampoGrande' in systems with and without incorporation of plant straw, respectively, compared with soybean succeeding maize(Vedoveto et al. 2013).Furthermore, in succession to Stylosanthes spp.'CampoGrande', soybean had an 85.31% lower P. brachyurus population than continuous soybean(Rodrigues et al. 2014).The results of the current study are relevant, as, in Brazil, P. brachyurus commonly infects soybean crops in high-production regions.Most of these elds are managed under no-till as a strategy to maintain soil health and increase crop yields.On the other hand, the majority of crops used in rotation with soybean are susceptible to P. brachyurus, such as maize, signalgrass, and millet(Debiasi etal.2016; Matias et al. 2018; Silva et al. 2018).Cultivation of Stylosanthes spp.'Campo Grande' before the main crop may contribute to reducing nematode populations.
(Lesturgez et al. 2004 et al. 2017al.2011representinga resistance mechanism of plants(Li et al. 2006;Medeiros et al. 2015; Ohri and Pannu  2010).Phenolic compounds also are as precursors of lignin and suberin(Espelie et al. 1986;Lyon et al. 1992), which are involved in the mechanical resistance of cells, reducing nematode penetration or movement(Ji et al. 2015).Proteins secreted by Stylosanthes 'Campo Grande' cells might also be involved in host resistance.Pathogenesis-related proteins are expressed during response to pathogen infection, interaction with bene cial microorganisms, and exposure to chemicals (Mauch-Mani et al. 2017).Many of these proteins are enzymes, such as phenylalanine ammonia-lyase, polyphenol oxidase, peroxidase, and β-1,3glucanase.These enzymes are associated with the synthesis of phytoalexins, phenols, and lignin, which affect nematode feeding and movement in plant tissues(Sankar et al. 2017;Seenivasan et al. 2011).The advantages of using Stylosanthes spp.are not limited to nematode control.The plant positively in uences soil properties, increasing fertility, total nitrogen, organic matter, and growth and activity of bene cial microorganisms(Long et al. 2007;Long et al. 2017).Furthermore, degradation of Stylosanthes spp.roots creates a large number of macropores in soil, promoting the development of subsequent plant roots(Lesturgez et al. 2004).