Two novel potential pathogens for soybean
Two novel potential pathogens for soybean
Binod Bihari Sahu, National Institute of
Technology Rourkela, INDIA
0 Department of Plant Protection, Instituto Agrono?mico do Parana ? , Londrina, Parana ? , Brazil
Nematode diseases have a worldwide importance for several economic agricultural crops, such as soybean. Frequently, new or secondary pathogens arise as emergent diseases due to the intensified use of agricultural lands, causing extensive yield losses. Helicotylenchus dihystera (Hd) and Scutellonema brachyurus (Sb) appear as potential pathogens for soybean in Brazil, since their spread and population densities have been increased on soybean growing areas. Aiming to evaluate the quantitative distribution of Hd and Sb in soybean fields in South Brazil, a survey was conducted during the growing seasons of 2014-2015 and 2015-2016 in which 1,088 soil samples and 1,043 root samples were analyzed. Besides, two greenhouse experiments were conducted to evaluate the pathogenicity of both nematodes to soybean plants, in comparison with P. brachyurus (Pb), a known pathogen of the crop. The survey demonstrated that Pb, Hd and Sb are widely distributed in the States of Parana? , Santa Catarina and Mato Grosso do Sul. Besides, we proved that Hd and Sb multiply and cause root lesions in soybean cv. Pot?ncia, since they were found inside roots, and can be considered as potential pathogens for soybean plants.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: The authors received no specific funding
for this work.
Competing interests: The authors have declared
that no competing interests exist.
The expansion of problems caused by nematodes in Brazil, especially in the Cerrado region,
probably is due to the intensified use of agricultural lands, with monoculture of susceptible
crops, which increase soil nematode populations along the crops. The economy in this region
is based largely on the viability of soybean, maize, and cotton [
Root-knot nematodes (RKNs) (Meloidogyne spp. Goeldi 1929) and the soybean cyst
nematode (SCN), Heterodera glycines Ichinoe, represent the most economically important
nematodes for soybean crop in Brazil and worldwide [
]. In the Cerrado region, after the impact
caused by H. glycines, two important changes occurred, the use of alternative plants for crop
rotation and the increase in the no-tillage system [
]. These changes directly affected
Pratylenchus brachyurus (Godfrey 1929) Filipjev & Sch. Stekhoven 1974 populations, which
currently is a concern as one of the main problems to soybean in Brazilian fields [
On soybean, yield losses caused by P. brachyurus can reach 30 to 50%, with higher damages
detected on sand soils (< 20% clay) [
]. Non-specific symptoms can be easily overlooked or
mistaken with damages caused by other soil pathogens or attributed to other causes such as
nutrient deficiency or drought [
Recently, other species, as Helicotylenchus dihystera (Cobb) Sher and Scutellonema
brachyurus Andrassy, appear as potential pathogens for soybean in Brazil, since their spread and
population densities have been increased on soybean growing areas [
]. The intensified use
of agricultural lands in Brazil could have a significant paper in the emerging of secondary
pathogens in agricultural crops .
Scutellonema brachyurus has been observed especially in the States of Parana?, Mato Grosso
do Sul and Maranh?o, in soybean fields showing stunting plants associated with yield losses
]. Soil nematode populations can reach up to 5,000 specimens per 100 cm3 of soil in samples
collected in soybean fields. Apparently, injuries are associated with drought and the use of
early cultivars [
Helicotylenchus dihystera, mainly associated with crop losses on sugarcane [
] and maize
], has also been detected in soybean growing areas in Brazil, with increasing incidences and
soil populations [
]. Surveys conducted in Acre, Mato Grosso, and Goia?s States showed that
H. dihystera was found in 85%, 92%, and 47% of the samples, respectively [
its presence has been associated with stunted plants, but direct damages were not quantified,
as well as the threshold levels.
The parasitism of H. dihystera in soybean plants was already reported by Orbin [
stained soybean roots infected with H. dihystera and observed adults, larvae and eggs within
the root cortex associated with small brown lesions in the immediate vicinity of the nematodes.
However, because of the lack of persistent burrows, the author suggested that H. dihystera was
not a significant pathogen of soybean.
In order to evaluate the quantitative distribution of H. dihystera and S. brachyurus in
soybean fields from the States of Parana?, Mato Grosso do Sul and Santa Catarina, a survey was
conducted during the growing seasons of 2014?2015 and 2015?2016 in which 1,088 soil
samples and 1,043 root samples were analyzed. Besides, two greenhouse experiments were
conducted to evaluate the pathogenicity of both nematodes on soybean plants, in comparison with
P. brachyurus, a known pathogen of the crop.
Materials and methods
Surveys were undertaken in commercial soybean fields in the States of Parana? (33
municipalities sampled), Mato Grosso do Sul (9 municipalities) and Santa Catarina (9 municipalities), in
the Southern Brazil (Fig 1), between October 2014 to April 2016. For that purpose, soybean
roots together with the rhizosphere (#1,043) and bulk soils (#1,088) were sampled. Samples
were collected with a hack from the upper 20 cm of soil of four to five plants chosen arbitrarily
in each field. All surveyed fields were representative of soybean growing areas in Southern
Brazil; they were selected based on indicative parameters of their commercial importance and of
previous problems with nematodes, according with the grower?s experience. Frequency of
infestation and population density of plant-parasitic nematodes were determined. The
frequency of infestation was calculated as the percentage of samples in which the nematode
species was diagnosed.
The entire root system was washed free of soil, weighed and used to extract nematodes by
the maceration procedure described in literature [
] without the use of sodium hypochlorite.
Nematodes present in the soil samples were extracted from a 50 cm3 subsample using the
Baermann funnel method [
]. Nematodes were counted and identified by morphological traits to
genus. Selected live adult female specimens of each genera were mounted on microscope slides
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Fig 1. Locations of soybean fields surveyed to determine the occurrence and distribution of Pratylenchus brachyurus,
Helicotylenchus dihystera and Scutellonema brachyurus in South Brazil.
with water for species identification. Pratylenchus brachyurus (Pb), Helicotylenchus dihystera
(Hd) and Scutellonema brachyurus (Sb) were identified through morphological and
morphometrical approaches, based on measurements of 10 adult females from each population [
Characters measured were: body length (L), stylet length (St), height (Hstk) and width
(Wstk) of stylet knobs, distance from posterior end to vulva (V), distance from vulva to anus
(V-A), tail length (T), post-uterine branch length (PUB), dorsal esophageal gland orifice
(DGO), maximum body diameter (BD), body diameter at anus (BDA) and vulva (BDV) to Pb;
L, V, lip height (LH), lip diameter (LD), St, conus length (CL), DGO, pharynx (P), excretory
pore (EP), BD, T, and BDA to Hd; and L, V, DGO, St, metenchium length (ML), telenchium
length (TL), P, EP, pharyngeal overlap (PO), BD, BDA, medium bulb length (MBL) and
diameter (MBD), LH, LD, lateral field width (LFW), T, scutellum length (SCL) and width (SCW),
and spermatheca length (SPL) and width (SPW) to Sb.
Besides, species delimitation was undertaken using an integrated approach considering
morphological and morphometric evaluation combined with molecular sequence analyses
]. For molecular analyses, specimens were picked individually from the water suspension
with the aid of a stereomicroscope and stored in saline solution (NaCl 1M). DNA from
individual females of each population was extracted using the methodology described in literature
]. ITS-1 and D2/D3 rDNA amplifications were performed using the universal primers 18S
(5?- TTG ATT ACG TCC CTG CCC TTT -3?) and RN58SR (5?- ACG AGC CGA
GTG ATC CAC CG -3?) to ITS-1 region, located respectively in the 18S and 5.8S rDNA
], and D2A (TCGGAAGGAACCAGCTACTA) and D3B (ACAAGTACCGTGAGGGAAAGTTG)
to D2/D3 region.
For PCR, 21 ?l of the master mix Platinum PCR Supermix (Invitrogen) were added to a 0.2
ml microcentrifuge tube; after, 2 ?l of DNA and 1 ?l of each primer were added.
Amplifications conditions for ITS-1 were as follows: 94 ?C for 2 min 45 s followed by 40 cycles of 94 ?C
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for 1 min, 57 ?C for 45 s and 72 ?C for 2 min, and a final extension of 72 ?C for 10 min; for D2/
D3: 94 ?C for 5 min followed by 35 cycles of 94 ?C for 30 s, 55 ?C for 45 s and 72 ?C for 2 min,
and a final extension of 72 ?C for 10 min. Purified DNA fragments were sequenced on a
sequencer ABI 377 DNA Sequencer and the quality of sequences was checked using the
software BioEdit. Detailed protocols were described by our colleagues [
] (Pb), [
] (Hd) and
] (Sb). The obtained D2/D3 sequences of each population species used for greenhouse
experiments were submitted to the GenBank database under accession numbers: EF693897
(Pb), MG365905 (Sb) and MG760573 (Hd).
Two greenhouse experiments were carried out at the Instituto Agrono?mico do Parana?,
Londrina, Parana? State, Brazil (23?18?36?S, 51?09?46?W). The trials were conducted from 11
November 2016 to 13 January 2017 with the temperature ranging from 22 ?C to 41 ?C.
Seeds of soybean cultivar BMX Pot?ncia were sown in 500 cm3 plastic pots containing 400
cm3 of sterilized (160?C/5hours) soil. Seedlings were thinned to one per pot prior to nematode
inoculation. Pratylenchus brachyurus population used as inoculum was collected in S?o Lu??s,
Maranh?o State, Brazil (02?31?48?S, 44?18?10?W), from soybean plants (Glycine max),
identified through morphological [
] and molecular [
] techniques and subsequently cultured
under greenhouse conditions in soybean cv. BMX Pot?ncia. Helicotylenchus dihystera and
Scutellonema brachyurus populations were collected in F?nix, Parana? State, Brazil (23?54?57?S,
51?58?44?W), both from soybean plants, identified through morphological and molecular
] and subsequently cultured under greenhouse conditions in soybean cv.
After nematode extraction from soybean roots by the maceration procedure described in
], without the use of sodium hypochlorite, a suspension containing 1,000 mixed
life stages (initial population, IP) was pipetted into two small 2.0?4.0 cm-deep holes beside the
root system of 5 days-old seedlings. Plants in pots were watered as needed and fertilized once
with 3 g of Osmocote1 Plus (15% N, 9% P2O5, 12% K2O, 1% Mg, 2.3% S, 0.05% Cu, 0.45%
Fe, 0.06% Mn, 0.02% Mo).
The final nematode population (FP) was evaluated 15, 30 and 70 days after inoculation
(DAI). At 15 and 30 DAI, plants were removed from pots and root systems were washed
carefully and stained with fuchsin acid [
]. The entire root system of each plant was examined
under light microscope. Besides, pots were immersed in a bucket containing 4 L of water to
separate roots from soil. This entire suspension was used to extract nematodes from the soil of
each replicate by Baermann funnel method [
]. At 70 DAI, roots were washed with tap water,
dried on absorbent paper, values of fresh weight determined and, subsequently, cut in 1 cm
pieces; the entire root system of each replicate was processed for nematode extraction, as
described earlier. Nematodes were also extracted from soil in this date [
]. FP was estimated
by counting mixed life stages from parasitized roots and soil with the aim of a Peter?s slide.
The reproductive factor (RF = FP (root+soil)/ IP) values were subsequently determined.
Number of nematodes per gram of roots (Nema/g) was also calculated for each replicate. In all
evaluations, fresh top weights were determined.
Experiments were arranged in completely randomized design, with treatments
corresponding to each nematode species and 8 replicates. Data were submitted to variance analysis and,
for data residuals normalization, BoxCox procedure indicated that data transformation by
ln(y+0.01) was necessary. Treatments were compared by Fisher?s Least Significant Difference
(LSD) Test (P 0.05), using the R 2.15.2 program [
], packages MASS [
] and Agricolae
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Fig 2. Frequency of occurrence of nematode species in the samples, highlighting the frequency of Pratylenchus brachyurus,
Helicotylenchus dihystera and Scutellonema brachyurus in soil (A) and root (B) samples.
Pb was found in 501 root samples (48.03%) and in 568 soil samples (52.21%), Hd in 300 root
(28.76%) and in 843 soil (77.48%) samples, and Sb in 164 root (15.72%) and in 328 soil
(30.15%) samples (Fig 2). The population densities of Pb ranged from 1 to 1,852 Nema/g and
from 1 to 1,060 nematodes per 50 cm3 of soil; for Hd, densities ranged from 4 to 168 Nema/g
and from 1 to 4,284 nematodes per 50 cm3 of soil, whereas to Sb densities ranged from 3 to
379 Nema/g and from 1 to 11,712 nematodes per 50 cm3 of soil. Generally, growers in these
localities described symptoms of stunting and chlorotic plants, associated with lower
productivities, but no quantifications were done, since other nematode species occurred in the
samples (Fig 2) and the isolation of damages caused by each one is impossible.
Except for slightly differences in tail shape, morphology of specimens of Pb, Hd and Sb
(Fig 3) compare very well with the previous descriptions of these species. In relation to
morphometric taxonomic characters of females, for P. brachyurus values measured, in ?m
(mean ? standard deviation), were: L = 543 (? 19.50), BD = 24 (? 2.20), BDV = 21 (? 1.43),
BDA = 15 (? 1.24), V% = 82 (? 4.55), St = 19 (? 0.81), Wstk = 4 (? 0.29), Hstk = 3 (? 0.44),
DGO = 2.78 (? 0.10), V-A = 54 (? 4.37), T = 28 (? 1.52), and PUB = 16 (? 2.41). For H.
dihystera females, values measured, in ?m (mean ? standard deviation), were:: L = 601 (? 44.30),
V = 209 (? 23.58), LH = 4 (? 0.32), LD = 7 (? 0.48), St = 26 (? 1.01), CL = 12 (? 0.86),
DGO = 13 (? 1.01), P = 142 (? 8.30), EP = 108 (? 6.94), BD = 25 (? 1.83), T = 16 (? 1.63), and
BDA = 16 (? 1.27). Finally, for S. brachyurus females, values measured, in ?m (mean ?
standard deviation), were:: L = 740 (? 30.06), V = 285 (? 18.67), DGO = 8 (? 0.62), St = 29 (?
1.23), ML = 14 (? 1.17), TL = 12 (? 1.11), P = 149 (? 8.03), EP = 116 (? 9.32), PO = 23 (? 2.39),
BD = 29 (? 1.27), BDA = 19 (? 0.72), MBL = 12 (? 0.78), MBD = 10 (? 0.56), LH = 6 (? 0.26),
LD = 9 (? 0.36), LFW = 4 (? 0.48), T = 4 (? 1.01), SCL = 5 (? 0.49), SCW = 4 (? 0.58), SPL =
11 (? 1.09), and SPW = 10 (? 0.92).
At 15 and 30 DAI, the observation of roots stained with fuchsin acid showed that the three
nematode species penetrated soybean roots cv. Pot?ncia (Figs 4 and 5). In experiment 1,
number of penetrated nematodes was lower than those found in experiment 2 (Fig 5A and 5B). At
15 DAI (Fig 5A), it was observed a lower number of nematodes inside roots than at 30 DAI
(Fig 5B), as expected.
In relation to the number of nematodes in soil (Fig 5C and 5D), we found a higher number
of exemplars of H. dihystera and S. brachyurus, in relation to P. brachyurus, at 15 DAI in soil,
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Fig 3. General morphology of females of Pratylenchus brachyurus (Pb), Helicotylenchus dihystera (Hd) and Scutellonema
brachyurus (Sb) populations from Brazil. A1) Sb entire body; A2) Sb anterior portion of body with labial region in evidence; A3)
Sb tail with circular scutellum in evidence; B1) Hd entire body; B2) Hd anterior portion of body with labial region in evidence; B3)
Hd tail; C1) Pb entire body; C2) Pb anterior portion of body with labial region in evidence; C3) Pb tail.
in both experiments (Fig 5C). Inversely, at 30 DAI, the number of P. brachyurus in soil was
higher than those from H. dihystera and S. brachyurus in experiments 1 and 2 (Fig 5D).
At 70 DAI, nematodes multiplied in soybean (Table 1), especially Hd and Sb, with RF values
higher than 1.0 in both experiments. In experiment 1, Hd showed the highest RF value in
soybean cv. Pot?ncia (RF = 2.33), followed by Sb (1.16); RF value for Pb was lower than 1.0 (0.63),
although this cultivar is reported to be susceptible to this root-lesion nematode species. In
relation to the number of nematodes per gram of roots, in experiment 1 we observed the lowest
number to Hd (29), probably as a consequence of the lowest value of FRW in this treatment.
In experiment 2, soybean plants inoculated with Hd showed the highest numbers of nema/g
and RF of this species. The total number of Pb found in soil (Table 1) was low in both
experiments, while Hd showed the highest values of SP. In relation to the total number of nematodes
Fig 4. Nematode-infected soybean roots stained with fuchsin acid. A) Pratylenchus brachyurus; B) Helicotylenchus dihystera; C)
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Fig 5. Number of nematodes inside roots of soybean cv. Pot?ncia. A and B) Experiments 1 and 2, respectively, 15 days after
inoculation; C and D) Experiments 1 and 2, respectively, 30 days after inoculation. Hd = Helicotylenchus dihystera; Pb =
Pratylenchus brachyurus; Sb = Scutellonema brachyurus.
in roots (Table 1), Pb and Sb showed the highest values in both experiments and Hd, the
In relation to the development of soybean plants inoculated with Pb, Hd and Sb at 70 dai
(Table 1), the FRW was significantly affected by nematodes in experiment 1, since FRW values
were lower than that obtained in non-inoculated plants. In this experiment, FRW was more
affected by Pb and Hd, with the lowest values, while plants infected with Sb showed
intermediate value of FRW. In experiment 2, the effect of nematodes in the development of soybean
roots was not observed, probably due to the better experimental conditions in this case.
For the variable FTW (Table 1), plants inoculated with Pb and Hd in experiment 1 showed
significantly lower values than those inoculated with Sb or the check plants. In experiment 2,
higher values of FTW were observed in plants inoculated with Hd and Sb, in relation to Pb
and check plants. This could had occurred due to the better development of roots in
experiment 2, which possibly allowed better development of the aerial parts of plants.
It was also observed a general darkening in the root systems of soybean cv. Pot?ncia
inoculated with Pb, Hd and Sb at 70 DAI, probably as a consequence of the coalescence of several
lesions caused by the nematodes (Fig 6). Symptoms attributed to Hd and Sb were very similar
to those reported for Pb.
In our study, we demonstrated that Pb, Hd and Sb are widely distributed in the States of
Parana?, Santa Catarina and Mato Grosso do Sul, as observed in another Brazilian soybean
growing regions [
]. Besides, we proved that Hd and Sb cause root lesions in soybean and can
be considered as potential pathogens for soybean plants.
In Goia?s State, Brazil, high population densities of Hd have been reported in corn growing
] and, posteriorly, it was observed that Hd was the prevalent nematode species in
coffee, corn and tomato growing areas in the State of Goia?s and in the Federal District [
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Each value represents the mean of 8 replicates. Means followed by the same letter in the column did not differ
according with the LSD test at 5% of significance.
aFresh root weight;
bFresh top weight;
cNumber of nematodes in soil + roots;
eNumber of nematodes per gram of roots.
Rio de Janeiro State, Hd was found in 30% of the samples collected in organic farms [
sugarcane fields from Pernambuco State, Hd was also the dominant species [
]. Hd was
the prevalent nematode species in forest fragments and agricultural areas cropped with corn
 and sugarcane [
] in Parana? State. In Mato Grosso State, it was observed that Hd was
present in 97% and 81% of the soil and root samples, respectively, collected in cotton growing
In relation to soybean, in Mato Grosso State, from 3,000 samples analyzed by the laboratory
from Fundac??o Rio Verde, Hd was found in 92% of them [
]. Hd was reported in 78% of the
samples collected in soybean fields in Rio Grande do Sul State [
] and in 47% of the samples
collected in Goia?s State [
]. Hd was also reported in 59% of the soybean samples collected in
Parana? State in a previous study [
]. Our results corroborate with the several reports of
prevalence of Hd in soil samples, since it was present in 77.48% of the collected samples, with
densities reached more than 4,000 nematodes per 50 cm3 of soil.
Sb has been found in different regions from the States of Parana?, Mato Grosso do Sul and
Maranh?o, with population densities higher than 5,000 per 100 cm3 of soil and even higher in
10 g of roots [
]. Sb also was found in 23% from the samples collected in soybean fields in
Parana? State in previous report [
]. In the present survey, Sb was found in 30.15% of the
samples and populations reached more than 11,000 nematodes per 50 cm3 of soil, higher than the
densities reported previously in soybean fields [
In relation to Pb, it was present in more than 50% of the soil samples in the present survey.
This species has become a concern among cotton and soybean growers in the Cerrado region
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Fig 6. Root lesions and darkening of soybean roots observed in plants inoculated with Pratylenchus brachyurus (Pb),
Scutellonema brachyurus (Sb) and Helicotylenchus dihystera (Hd). From left to right: check (without nematode), Pb, Sb and Hd.
and nowadays is considered the main nematode species to soybean in Brazil [
soybean, losses caused by this nematode can reach to 30 to 50% of the yield, in which higher
damages are observed in sand soils (< 20% clay) . Symptoms are stunting of plants and leaf
yellowing, besides the root lesions, characteristics from this genus [
]. We observed stunted
and chlorotic plants, associated with lower productivities in the surveyed fields, but no
quantifications or correlations with the nematode densities were done.
As observed previously [
], Hd can be found inside the soybean roots, causing brown
lesions, contradicting the idea that this nematode is a migratory ectoparasitic or
semi-endoparasitic species that may occur in very high numbers only in soil surrounding host roots [
Indeed, the majority of studies reported only the association of Hd with several plant species,
including soybean, but no symptoms or damages have been quantified [
41, 42, 38, 43
general darkening of roots observed in our study configurate a more aggressive symptom than
those previously reported , confirming the hypothesis that Hd, together with Sb, can be
considered as a potential pathogen to soybean in Brazil.
Similarly, Sb has been considered as a primarily ectoparasitic [
] associated with a range
of agricultural and horticultural crops [
]. In our study, we observed the penetration of this
species in soybean roots, as observed for Hd. Reports of the parasitism or association between
Sb and soybean are scarce in worldwide literature [
], but in some regions of Brazil, as Parana?
State, this nematode has been frequently found in soil and root samples collected in soybean
Both nematodes could facultatively had developed the migratory endoparasitic habit in
order to better adaptation in hosts used in a cropping system, as the intensified use of
agricultural lands provide roots along the year for nematode parasitism and adaptation. As an
example, the evolution of plant parasitism in nematodes is reported for Aphelenchoides spp., which
feed and reproduce on plant leaf mesophyll and on buds (as a migratory endoparasite) but can
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also feed on fungi [
]. Recently, this group was reported as the causal agent of a new soybean
disease in Brazil, the soybean green stem and foliar retention syndrome [
], and also causing
similar symptoms in cotton plants [
], which shows the great plasticity of Aphelenchoides spp.
in adaptation to hosts imposed in the cropping systems. The same plasticity could be suggested
to exist in Hd and Sb.
Since Hd and Sb were found inside soybean roots, root lesions and generalized darkening
of root systems were observed, as a result of this type of parasitism (as showed in Fig 6). Root
lesions caused by Sb in soybean were not yet reported in literature. Although losses were not
quantified, it is expected to occur a reduction in soybean productivity when it is cropped in
infested areas with Hd and Sb, as observed for Pb, since both penetrated and caused damages
on roots, multiplied on soybean, and, therefore, considering their widespread, can be
considered as potential pathogens for this crop and possibly to others, which comprise the cropping
systems in Brazil.
S1 File. Data analysis for experiment 1, evaluation at 15 days after inoculation.
S2 File. Data analysis for experiment 2, evaluation at 15 days after inoculation.
S3 File. Data analysis for experiment 1, evaluation at 27 days after inoculation.
S4 File. Data analysis for experiment 2, evaluation at 27 days after inoculation.
S5 File. Data analysis for plant development in experiment 1, evaluation at 70 days after
S6 File. Data analysis for nematode multiplication in experiment 1, evaluation at 70 days
S7 File. Data analysis for plant development in experiment 2, evaluation at 70 days after
S8 File. Data analysis for nematode multiplication in experiment 2, evaluation at 70 days
Conceptualization: Andressa Cristina Zamboni Machado, Santino Aleandro da Silva.
Data curation: Andressa Cristina Zamboni Machado, Priscila Moreira Amaro, Santino
Aleandro da Silva.
Formal analysis: Andressa Cristina Zamboni Machado, Santino Aleandro da Silva.
Investigation: Priscila Moreira Amaro, Santino Aleandro da Silva.
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Methodology: Andressa Cristina Zamboni Machado, Priscila Moreira Amaro, Santino
Aleandro da Silva.
Project administration: Andressa Cristina Zamboni Machado.
Resources: Andressa Cristina Zamboni Machado.
Software: Santino Aleandro da Silva.
Supervision: Andressa Cristina Zamboni Machado.
Validation: Priscila Moreira Amaro, Santino Aleandro da Silva.
Writing ? original draft: Andressa Cristina Zamboni Machado.
Writing ? review & editing: Andressa Cristina Zamboni Machado, Priscila Moreira Amaro,
Santino Aleandro da Silva.
11 / 13
12 / 13
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