Archives of Microbiology (2023) 205:209

https://doi.org/10.1007/s00203-023-03555-3

ORIGINAL PAPER

Rhizobacteria control damping‑off and promote growth of lima bean

with and without co‑inoculation with Rhizobium tropici CIAT899
Linnajara de Vasconcelos Martins Ferreira1,2 · Rafael de Almeida Leite1 · Fernanda de Carvalho1 ·
Júlia Fonseca Colombo Andrade1 · Flávio Henrique Vasconcelos de Medeiros3 · Fatima Maria de Souza Moreira1

Received: 13 November 2022 / Revised: 14 April 2023 / Accepted: 15 April 2023 / Published online: 27 April 2023
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2023

Abstract
Rhizoctonia solani compromises the production of lima bean, an alternative and low-input food source in many tropical
regions. Inoculation of bacterial strains has been used, but research on their biocontrol and growth promotion potential
on lima bean is scarce. The objective of this study was to evaluate the effects of inoculation with rhizobacterial strains of
the genera Bacillus, Brevibacillus, Paenibacillus, Burkholderia, Pseudomonas, and Rhizobium in combination or not with
­N2-fixing Rhizobium tropici on the control of damping-off disease and growth promotion in lima bean plants. Greenhouse
experiments were conducted to evaluate the inoculation with bacterial strains with biocontrol potential in combination or
not with R. tropici in substrate infected with R. solani CML 1846. Growth promotion of these strains was also assessed.
Strains of Brevibacillus (UFLA 02-286), Pseudomonas (UFLA 02-281 and UFLA 04-885), Rhizobium (UFLA 04-195),
and Burkholderia (UFLA 04-227) co-inoculated with the strain CIAT 899 (Rhizobium tropici) were the most effective in
controlling R. solani, reducing the disease incidence in 47–60% on lima bean. The promising strains used in the biocontrol
assays were also responsive in promoting growth of lima bean under disease and sterile conditions. A positive synergistic
effect of co-inoculation of different genera contributed to plant growth, and these outcomes are important first steps to
improve lima bean production.

Keywords  Phaseolus lunatus · Rhizoctonia solani · Thanatephorus cucumeris · Rhizoctoniosis · PGPR · Biocontrol

Introduction prominent producers with over 12,000 hectares cultivated

(USDA 2019). In Brazil, lima bean is grown by small farm-
Lima bean (Phaseolus lunatus L.) is the second most com- ers mainly in the Northeastern region, under semi-arid cli-
mercialized crop from the genus Phaseolus (Fofana et al. mate as a subsistence crop. Yield and technological inputs
1999). Worldwide, lima bean shares the importance of legu- are usually low, but there is always a demand for lima bean
minous crops with soybean, common beans, and cowpea as an alternative protein source (Alves et al. 2008). The crop
as direct plant protein source. The USA is one of the most is severely affected by different groups of the Rhizoctonia
complex, and symptoms are diverse (Assunção et al. 2011;
Yang and Li 2012).
Communicated by Yusuf Akhter. Damping-off caused by the fungus Rhizoctonia solani
Kuhn is the most substantial cause of root disease in the
* Fatima Maria de Souza Moreira world for several crops, such as maize, rice, wheat, soybean,
[email protected]
peanut, dry bean, potato, cotton, etc. (Lamichhane et al.
1
Departamento de Ciência Do Solo, Setor de Biologia, 2017; Tziros and Karaoglanidis 2022). R. solani severely
Microbiologia E Processos Bioquímicos Do Solo, compromises the production of beans from the genus Pha-
Universidade Federal de Lavras, UFLA, C.P. 3037, Lavras, seolus. The damage caused by R. solani occurs up to three
MG 37200‑900, Brazil
weeks after emergence, and symptoms include seed and
2
Instituto Federal Do Pará, IFPA, Campus Marabá Rural, root rot, stem canker, leaf blight, and seedling damping-off
C.P. 041, Marabá, PA 68508‑979, Brazil
(Ajayi-Oyetunde and Bradley 2018).
3
Departamento de Fitopatologia, Universidade Federal de
Lavras, UFLA, C.P. 3037, Lavras, MG 37200‑900, Brazil

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Control of R. solani can be very difficult due to its viru- R. solani damping-off (Jung et al. 2003; Quan et al. 2006;
lence, a wide host range, transmission through seeds, and Leite et al. 2013). Furthermore, the literature also reports
overwintering as a saprotroph in the crop stubble or as scle- that the biocontrol effect of PGPR can be enhanced when
rotia (Ajayi-Oyetunde and Bradley 2018). In many regions in co-inoculation with species of Rhizobium, including R.
of the world, to prevent disease losses, lima bean farmers tropici (Jensen et al. 2002; Kalantari et al. 2018). The mech-
leave the infected areas, which has a significant economic anisms promoted by these bacterial genera are diverse and
impact and depreciates the land. The traditional control include production of antibiotics, bacteriocins, siderophores,
methods, such as the use of more resistant cultivars, fun- enzymes, plant hormones, and others (Jiao et al. 2021).
gicides, and crop rotation, might reduce the soil pathogen We hypothesized that 15 strains with growth promotion
population but may not be satisfactory to control the disease and damping-off control abilities that were promising in
or its adoption is rare for faba bean as it is for other legumes common beans would also be important biological assets
and pulses. Thus, other methods should be combined to keep for the disease control in lima bean since these plants are
R. solani below the damage threshold level (Lamichhane from the same genus. Furthermore, co-inoculation of Rhizo-
et al. 2017). In Brazil, the registration of biocontrol products bium tropici CIAT899 with these strains could promote a
for plant disease management is based on the target patho- stronger biocontrol activity, as previously shown in other
gen and not the crop. Some strains of Thrichoderma spp., works for common-bean (Jensen et al. 2002; Ferreira et al.
Bacillus pumillus, and Burkholderia cepacia are available as 2020). Therefore, the objective of this study was to evaluate
commercial products (Bettiol et al. 2012).  Therefore, there the effects of inoculation with 15 rhizobacterial strains of the
is no product specifically registered for lima bean damping- genera Bacillus, Brevibacillus, Paenibacillus, Burkholderia,
off caused by R. solani and there is no scientific endorsement Pseudomonas, and Rhizobium in combination or not with
with peer-reviewed publication for this matter. The selection Rhizobium tropici CIAT899 on the control of damping-off
of lima bean genotypes resistant to damping-off is also still a disease and on growth promotion in lima bean plants.
challenge, and the search for alternative methods is essential
to limit disease damage, reduce the density of the pathogen
population in the soil, and increase crop yield. There are no Materials and methods
currently available lima bean cultivars that are resistant to
R. solani in Brazil. Preparation of microbial inocula
The use of plant growth-promoting rhizobacteria (PGPR)
has been suggested as a biological control for damping-off In this study, 15 bacterial strains selected from the collec-
(Noronha et al. 1995; Martins et al. 2018) and is used in tion of the Soil Microbiology and Biological Processes
Brazil as part of integrated disease management on other Laboratory of the Universidade Federal de Lavras, Bra-
crops, such as common bean. For common bean plants, stud- zil, were used (Table 1). These strains had been isolated
ies show that it is possible to control damping-off disease from different sites and land use systems in Brazil and were
with PGPR that also have positive effects on nodulation and tested for several growth promotion traits in vitro and on
N fixation (Elkoca et al. 2010; Martins et al. 2018; Ferreira common bean, cowpea, and siratro. The Rhizobium tropici
et al. 2020). The synergistic PGPR effects are especially strain CIAT 899 was also used in the experiments. Rhizo-
advantageous considering organic, agroecological, and low- bium tropici CIAT 899 is a genetically stable N
­ 2-fixing strain
input production systems that do not use mineral N or syn- adapted to tropical conditions and tolerant to abiotic stress
thetic fungicides. However, to the best of our knowledge, conditions, such as high temperatures and acidity (Martínez-
there is no report on control of damping-off with PGPR or Romero et al. 1991; Graham 1992). The CIAT 899 strain
on plant growth promotion mediated by beneficial bacteria has been approved by the Brazilian Ministry of Agriculture
for lima bean. as an inoculant for common bean; however, it is not able to
Previous studies from our laboratory identified several establish ­N2-fixing symbiosis with lima bean. All bacterial
strains of different bacterial genera with various growth strains were cultured separately in liquid 79 medium con-
promotion traits (Ferreira et al. 2012, 2018; Costa et al. ­ −1): ­K2HPO4, 0.5; M
taining (g L ­ gSO4.7H2O, 0.2; NaCl, 0.1;
2013). Some strains were tested previously for control of mannitol, 10.0, and yeast extract, 0.4; pH 6.8–7.0 (Fred and
damping-off in common bean, and strains of the genera Waksman 1928). The inocula were cultivated at 28 °C under
Pseudomonas and Brevibacillus were considered promis- shaking at 110 rpm for 3 days for fast-growing strains and
ing (Ferreira et al. 2020). Other studies also report these 7 days for slow-growing strains. Bacterial dose was applied
two genera for control of damping-off disease in common at ­108–9 CFU ­ml−1.
bean (Martins et al. 2018) and cowpea (Noronha et al. 1995). Rhizoctonia solani CML 1846 was provided by the Myco-
Strains from other genera, such as Bacillus, Burkholderia, logical Collection of the Universidade Federal de Lavras.
and Paenibacillus, have also been reported as biocontrol of We have used the Anastomosis group 4, which has a broad

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Table 1  Identification and origin of the strains

Strain Species Origin/land ­usea Growth charac- Accession number References
teristics in 79 in GenBank (NCBI)
medium
GRb pH

UFPI B3-9 Paenibacillus sp. PI/AGRI Fast Acidic KF738126 Costa et al. (2013)
UFPI B4-9 Paenibacillus sp. PI/AGRI Fast Alkaline KF738122 Costa et al. (2013)
UFLA 02-281 Pseudomonas sp. AM/P Fast Acidic KU613382 Ferreira et al. (2018)
UFLA 02-286 Brevibacillus sp. AM/P Fast Acidic KU613387 Ferreira et al. (2020)
UFLA 02–290 Bacillus megaterium AM/P Fast Acidic KU613390 Ferreira et al. (2018)
UFLA 02-293 Pseudomonas putida AM/P Fast Acidic KU613392 Ferreira et al. (2018)
UFLA 03-10 Paenibacillus kribbensis MG/SDTF Fast Acidic JQ041885 Marra et al. (2012)
UFLA 03–107 Bacillus subtilis MG/SDTF Fast Acidic JQ041895 Marra et al. (2012)
UFLA 03-18 Pseudomonas sp. AM/SF Fast Acidic KC879697 Oliveira-Longatti et al. (2014)
UFLA 03–26 Pseudomonas sp. AM/P Fast Acidic KC879702 Oliveira-Longatti et al. (2014)
UFLA 04–122 Burkholderia fungorum AM/PF Fast Acidic JF412046 Ferreira et al. (2012)
UFLA 04–195 Rhizobium miluonense AM/FA Fast Acidic JF412048 Ferreira et al. (2012)
UFLA 04–227 Burkholderia fungorum AM/AGRI Fast Neutral JF412051 Ferreira et al. (2012)
UFLA 04–885 Pseudomonas koreeensis RO/P Slow Alkaline KC879711 Oliveira-Longatti et al. (2014)
629 Bacillus subtilis BA/AGRI JQ435867 Leite et al. (2013)
CIAT 899 Rhizobium tropici Colombia Fast Acidic – Martínez-Romero et al. (1991)
a
 States of AM Amazônia, MG Minas Gerais, PI Piauí, RO Rondônia, AGRI agriculture, P pasture, PF primary forest, SF secondary forest, FA
secondary forest in advanced stage of regeneration, SDTP semi-deciduous tropical forest
b
 GR growth rate, fast: 2–3 days, slow: 6–10 days

host range and is the most ubiquitous one. The inoculum was Two greenhouse experiments with the same treatments
prepared in flasks containing 100 g of autoclaved hulled rice were conducted at the Universidade Federal de Lavras
substrate (120 °C, 30 min, 101 kPa) and 40 mL of sterilized (21.2275° S, 44.9781° W) in a Latossolo Vermelho distrofér-
distilled water. Each flask received a 5-mm-diameter disk rico soil (Oxisol, in approximation to the taxonomy of the
of the fungal culture, previously cultured in potato dextrose United States Department of Agriculture) to test the efficacy
agar medium (200 g L ­ −1 potato infusion, 20 g L
­ −1 dextrose, of the 15 bacterial strains against R. solani on lima bean. The
−1
and 17 g L­ agar) for 7 days. After 10 days of incubation at soil (pH = 5.6; N = 1,030 mg ­kg−1; P = 3 mg ­kg−1; K = 0.14
25 °C, the colonized substrate was placed in paper bags and ­cmolc ­dm−3; Al = 1.40 ­cmolc ­dm−3; Ca + Mg = 0.80 ­cmolc
dried for 48 h before being ground in a blender (Noronha ­dm−3) was mixed with washed sand at a 2:1 (soil:sand) ratio.
et al. 1995). The substrate was autoclaved twice at 121 °C and 147 kPa
for 1 h. After 15 days, the mixture was placed in 0.5-L plas-
tic pots and the moisture was adjusted to 60% of field capac-
Efficiency of bacterial strains for R. solani control ity. The substrate was inoculated with R. solani CML 1846
in lima bean using the inoculum described above 24 h before sowing at
a dose of 50 mg ­kg−1. A previous test with the inoculum
The experiment to test the biocontrol of R. solani by the ensured that all plants were infected by the pathogen.
PGPR strains was carried out in a completely randomized Seeds of lima bean variety Rajada, obtained in Floriano,
experimental design with four replicates and the following Piauí, Brazil, were surface disinfected using 70% ethanol for
treatments: single inoculation with the 15 PGPR strains; co- 30 s and 2% sodium hypochlorite for 2 min. After disinfec-
inoculation of the 15 PGPR strains with R. tropici CIAT tion, the seeds were thoroughly washed in sterilized distilled
899; CIAT 899 inoculated alone; and a control without water. Four seeds were sown for each pot (4 pots per treat-
inoculation. The strain 629 of Bacillus subtilis was used as ment) and each seed received 1 mL of bacterial inoculant,
a positive control since it was previously identified as an according to the respective treatment. The non-inoculated
antagonistic strain (Leite et al. 2013). The experiment was control treatment received 1 mL of sterilized water. Pots
repeated 30 days afterward under the same conditions. were constantly irrigated to maintain field capacity at 60%.

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The temperature inside the greenhouse ranged from 22 to strains UFLA 02-281, UFLA 02-286, UFLA 04-195, UFLA
25 °C for the first trial and 24–27 °C for the second. 04-227, and UFLA 04-885, with CIAT 899, plus the inocula-
Lima bean plants were collected at second true leaf stage tion of CIAT 899 alone and five pathogen doses (0, 50, 100,
(approximately 27 days after sowing), air-dried, and evalu- 150, and 200 mg ­kg−1). Four replications were carried out.
ated for plant height (PH); shoot, root, and total dry weight Substrate, microbial inocula, and seeds were prepared as
(SDW, RDW, and TDW); number of nodules (NN); nod- previously described. Five seeds were sown in 0.5-L pots
ule dry weight (NDW); germination rate (GR); and disease with autoclaved substrate (2:1 mixture of soil:sand) with the
severity, according to Noronha et al. (1995). Disease severity respective dose of the pathogen inoculum. After emergence,
was measured every 3 days over 15 days after emergence pots were thinned to two plants. Plants were evaluated as
based on a 0 to 4 scale, where 0 = no symptoms; 1 = small described in the previous experiment. Data were assessed for
lesions on the hypocotyl; 2 = large lesions on the hypoco- normality and homoscedasticity of the residues, by analysis
tyl; 3 = severely damaged hypocotyl, damping-off; 4 = non- of variance, and by polynomial regression for the pathogen
germinated seeds. The disease severity was transformed to inoculum dose and the disease index (DI).
a disease index (DI), according to McKinney (1923), and
used to calculate the area under the disease progress curve Plant growth promotion of lima bean
(AUDPC), according to the equation (Shaner and Finney under different N supply
1977):
n The strains used in this work were previously tested for
growth promotion on other crops, such as common bean. We
∑ [( ) ]
AUDPC = Yi+1 + Yi ∕2 [Xi+1 + Xi ]
i=1 hypothesized that they could also be able to promote growth
of lima bean plants under axenic conditions and different N
In which ­Yi = disease severity (per unit) at the ith obser- concentrations/supply, following the arrangement described
vation; ­Xi = time (days) at the ith observation, and n = total in Ferreira et al. (2020). A completely randomized experi-
number of observations. mental design was used in a 15 × 3 factorial arrangement.
All data were assessed for normality and homoscedas- The first factor consisted of inoculation of the 14 PGPR
ticity of the residues and by analysis of variance. The two strains plus a control without inoculation. The second fac-
replications of the test were compared by the F-test since tor was the supply of N, composed of inoculation with the
there were only two factors. All variables were presented CIAT 899 strain of R. tropici, a low mineral N concentration
as means of the two repetitions of the experiment. Since of 5.25 mg ­L−1 (LN), and a high mineral N concentration of
the repetitions were performed 30 days apart, environmen- 52.5 mg ­L−1 (HN). The treatments that received CIAT 899
tal conditions could be the cause of differences in some also received 5.25 mg L ­ −1 of N. Different mineral N con-
variables. In this case, the means of both repetitions could centrations were provided through a nutrient solution using
approach a more realistic condition instead of just one set of ­KNO3 and Ca(NO3)2 as sources of N (Hoagland and Arnon
data. Treatments were then compared by analysis of variance 1950). Three replications were carried out.
and means were grouped by the Scott–Knott test (p < 0.05). The experiment was conducted in Leonard jars containing
A principal component analysis (PCA) for the biocontrol a 1:1 (v:v) mixture of sand and vermiculite as substrate and
experiment was performed with the means of the variables the nutrient solution. The jars were autoclaved at 121 °C
to calculate the scores. Results were presented in the form and 147 kPa for 1 h. Four surface-sterilized lima bean seeds
of biplots. The PCA was calculated with the package vegan were sown in each jar, and each seed received 1 mL of the
(Oksanen et al. 2019). Statistical analyses were carried out in respective bacterial inoculant according to the treatments.
Sisvar 5.7 software (Ferreira 2011), R environment (R Core After sowing, the jars were covered with a layer of paraffin
Team 2021), and the R Studio platform (RStudio 2021). sand (mixture of 10 kg of washed sand, 1 mL of chloroform,
and 10 g of paraffin) to avoid contamination. After emer-
Severity of damping‑off in relation to R. solani gence, seedlings were thinned to leave one plant per jar. The
inoculum density in lima bean nutrient solution was replenished periodically throughout
the experiment.
The objective of the following experiment was to test the Plants were harvested at the beginning of the flowering
biocontrol effect of bacterial strains depending on the stage, approximately 40 days after sowing. Shoots and roots
pathogen density of R. solani CML 1846. Strains with the were air dried; and shoot, root, and total dry weight were
best biocontrol effect observed in the first experiment were assessed (SDW, RDW, and TDW, respectively). Shoots were
included in this experiment. A completely randomized then ground, and N concentration was determined by the
experimental design was used in a 6 × 5 factorial arrange- semi-micro-Kjeldahl method (Liao 1981). Shoot N accu-
ment. The factors consisted of five co-inoculations of mulation (SNA) was calculated as SDW × N concentration.

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Statistical analyses were carried out in Sisvar 5.7 software co-inoculations stood out, the other treatment scores were
(Ferreira 2011), R environment (R Core Team 2021), and the scattered throughout the components. Interestingly, the other
R Studio platform (RStudio 2021). Data were evaluated for co-inoculations were positively related to the PC1 and the
normality and homoscedasticity of the residues, and analysis disease indices, while some single inoculations were not.
of variance and means were compared by the Scott–Knott The control that did not receive any inoculation was closely
test (p < 0.05). A PCA was performed for the plant growth related with the disease indices and negatively related to all
promotion experiment with the means of the variables. other variables.
Results were presented in the form of biplots. The PCA was
calculated with the package vegan (Oksanen et al. 2019). Severity of damping‑off in relation to R. solani
inoculum density in lima bean

Results There was no evidence of damping-off in lima bean plants

when the pathogen was not inoculated in the substrate. When
Efficiency of bacterial strains for R. solani control plants were grown in the substrate with different doses of R.
in lima bean solani, plants with the UFLA 02-286 + CIAT 899, UFLA
04-195 + CIAT 899, UFLA 04-227 + CIAT 899, and UFLA
Most of the treatments led to an increase in the germination 04-885 + CIAT 899 treatments were able withstand the path-
rate (GR) of the seeds compared to the control without inoc- ogen at the dose of 50 mg ­kg−1 than plants in the treatment
ulation (Table 2). The inoculations UFLA 02-281, UFLA with CIAT 899 alone (Fig. 2). At the dose of 100 mg ­kg−1,
02-286, and UFLA 02-286 + CIAT 899 resulted in 100% plants co-inoculated with UFLA 04-195 + CIAT 899, UFLA
seed germination. Several co-inoculations led to superior 04-227 + CIAT 899, and UFLA 04-885 + CIAT 899 showed
results for plant height (PH) in comparison with plants inoc- moderate pathogen management. At the highest pathogen
ulated with the efficient CIAT 899 strain alone (Table 2). doses, 150 and 200 mg ­kg−1, none of the treatments were
TDW and SDW followed the same pattern, with almost the able to reduce the disease, and the DI approached 100%.
same treatments accumulating more biomass than the treat- Although they did not perform as much as the other treat-
ment with CIAT 899 alone (Table 2). Only plants inoculated ments, inoculations with UFLA 02-281 + CIAT 899 and
with UFLA 02-293 were similar to the non-inoculated con- CIAT 899 alone were able to promote some disease man-
trol. For RDW, several treatments led to an increase in root agement at the lowest inoculum dose (50 mg ­kg−1).
biomass compared to the control, including inoculation with
CIAT 899. Plant growth promotion of lima bean
All treatments reduced the disease index (DI) in com- under different N supply
parison with the non-inoculated control (Table 2). Greater
significant reductions in DI and AUDPC were found in the Inoculation of the PGPR strains on lima bean plants showed
treatments UFLA 02-281, UFLA 02-286, UFLA 04-195, diverse responses in the three forms of N supply (Table 3).
UFLA 04-885, and UFLA 04-227, all co-inoculated with Inoculation with UFLA 02-290, UFLA 02-293, UFLA
CIAT 899. These treatments reduced the DI by up to 60%. 03-107, and UFLA 04-885 led to similar SDW in all the
The PCA (Fig. 1) confirms these results as these co-inoc- forms of N supply. Plants inoculated with UFPI B3-9 had
ulations clustered and showed positive correlation with the greater SDW than plants that received the low N concentra-
plant-growth variables (except RDW), while being oppo- tion (LN). In contrast, plants inoculated with UFLA 03-18
site to the disease variables. Other treatments, such as UFPI developed more SDW in the LN than when co-inoculated
B4-9 + CIAT 899, UFLA 03-10, UFLA 04-122, also exhib- with CIAT 899. All other inoculations were more responsive
ited low values of AUDPC (Table 2). All treatments showed when associated with a high N concentration (HN). Within
lower AUDPC than the control, except for the UFLA 02-290 each form of N supply, responses varied according to the
and UFLA 02-290 + CIAT 899 treatments. There was a strain inoculated. UFLA 02-286, UFLA 02-293, UFLA
strong correlation between DI and AUDPC as showed by 04-195, and UFLA 04-227 promoted greater shoot growth
the PCA and, generally, treatments that showed low DI also when co-inoculated with CIAT 899 and in LN concentra-
showed low AUDPC. tion than the controls inoculated with CIAT 899 alone and
The PCA (Fig. 1) also shows positive correlations among the non-inoculated control. Among the plants fertilized with
SDW, TDW, and PH, while germination and RDW were low HN concentration, the best shoot growth was promoted by
or not correlated with these variables. As expected, DI and UFLA 03-18. For RDW, UFLA 02-286, UFLA 02-293,
AUDPC were negatively correlated with the plant growth UFLA 03-10, UFLA 04-227, and the control did not differ
variables. It is presumed that higher disease incidence on the among the forms of N supply. The other inoculations accu-
plants prevents their growth. Despite the abovementioned mulated more root dry weight when supplied with HN. In

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Table 2  Germination (GERM), GERM PH TDW SDW RDW DIa AUPDCa

plant height (PH), total dry
weight (TDW), shoot dry % cm g ­plant−1 %
weight (SDW), root dry weight
Experiment ­repetitionsb
(RDW), disease index (DI), and
area under the disease progress  1st 81.9 5.05 0.37* 0.26* 0.10* 71.03 4205.7
curve (AUDPC) of lima bean  2nd 80.8 5.29 0.29* 0.23* 0.07* 73.37 4321.7
plants inoculated with PGPR Treatmentc
and co-inoculated with PGPR
 UFPI B3-9 60.0 e 3.97 d 0.52 a 0.41 a 0.10 a 66.72 d 3925.7 e
and Rhizobium tropici CIAT
899 grown on soil infected with  UFPI B3-9 + CIAT 899 65.0 e 5.63 b 0.39 b 0.27 b 0.12 a 88.52 b 4333.4 d
Rhizoctonia solani   UFPI B4-9 77.5 d 4.41 d 0.48 a 0.35 a 0.13 a 68.52 d 4659.5 c
 UFPI B4-9 + CIAT 899 83.1 c 6.88 a 0.45 a 0.35 a 0.10 a 85.50 c 3328.2 f
 UFLA 02-281 100.0 a 5.45 b 0.39 b 0.33 a 0.06 b 79.88 c 4132.1 d
 UFLA 02-281 + CIAT 899 75.0 d 6.03 a 0.45 a 0.39 a 0.06 b 42.35 g 3431.8 f
 UFLA 02-286 100.0 a 4.98 c 0.36 b 0.32 a 0.05 b 82.54 c 4563.6 c
 UFLA 02-286 + CIAT 899 100.0 a 4.38 d 0.44 a 0.37 a 0.07 b 35.83 g 4651.7 c
 UFLA 02-290 85.0 c 4.35 d 0.23 c 0.16 c 0.06 b 83.25 c 5457.9 a
 UFLA 02-290 + CIAT 899 80.0 c 4.65 c 0.32 c 0.26 b 0.06 b 81.67 c 5423.0 a
 UFLA 02-293 65.0 e 5.10 c 0.16 d 0.10 d 0.06 b 82.75 c 4722.1 c
 UFLA 02-293 + CIAT 899 65.0 e 5.41 b 0.30 c 0.20 c 0.10 a 70.63 d 5225.7 b
 UFLA 03-10 95.0 b 4.76 c 0.25 c 0.17 c 0.08 b 71.83 d 3437.4 f
 UFLA 03-10 + CIAT 899 80.0 c 4.46 d 0.28 c 0.17 c 0.11 a 73.79 d 4296.5 d
 UFLA 03-107 92.5 b 5.44 b 0.32 c 0.26 b 0.06 b 83.89 c 4543.1 c
 UFLA 03-107 + CIAT 899 75.0 d 4.23 d 0.30 c 0.21 c 0.09 a 62.08 e 4325.9 d
 UFLA 03-18 80.0 c 5.00 c 0.24 c 0.15 c 0.08 b 84.67 c 3108.5 f
 UFLA 03-18 + CIAT 899 65.0 e 5.18 b 0.31 c 0.18 c 0.13 a 80.52 c 4245.1 d
 UFLA 03-26 80.0 c 5.43 b 0.40 b 0.31 b 0.10 a 76.54 d 4523.6 c
 UFLA 03-26 + CIAT 899 80.0 c 5.00 c 0.34 b 0.21 c 0.13 a 71.33 d 4375.9 d
 UFLA 04-122 85.0 c 5.20 b 0.35 b 0.28 b 0.06 b 66.79 d 3137.1 f
 UFLA 04-122 + CIAT 899 82.5 c 6.11 a 0.25 c 0.17 c 0.07 b 83.54 c 4960.3 b
 UFLA 04-195 92.5 b 5.60 b 0.35 b 0.28 b 0.07 b 84.10 c 4562.1 c
 UFLA 04-195 + CIAT 899 95.0 b 6.10 a 0.41 b 0.32 a 0.09 a 38.96 g 3531.5 f
 UFLA 04-227 65.0 e 5.55 b 0.27 c 0.21 c 0.06 b 80.63 c 4316.5 d
 UFLA 04-227 + CIAT 899 92.5 b 6.40 a 0.44 a 0.33 a 0.10 a 48.54 f 2347.8 g
 UFLA 04-885 83.8 c 4.58 c 0.28 c 0.18 c 0.10 a 87.29 b 4244.8 d
 UFLA 04-885 + CIAT 899 98.8 a 4.63 c 0.45 a 0.36 a 0.08 b 40.83 g 3545.2 f
 629 85.0 c 5.70 b 0.23 c 0.15 c 0.08 b 81.42 c 4326.5 d
 629 + CIAT 899 85.8 c 5.83 b 0.24 c 0.15 c 0.08 b 69.17 d 4744.0 c
 CIAT 899 75.0 d 5.25 b 0.37 b 0.25 b 0.12 a 60.69 e 4455.9 c
 Control 60.0 e 3.95 d 0.12 d 0.07 d 0.05 b 95.67 a 5556.3 a

Means of the treatments refer to the mean of the two repetitions of the experiment for all variables
a
 Higher values indicate lower plant resistance to Rhizoctonia solani
b
 Mean values followed by * in the column differ by the F test (P < 0.05) for the experiment replications
c
 Mean values followed by the same letter in the column do not differ by the Scott–Knott test (P < 0.05) for
the treatments

addition, there was no difference in RDM within each form with UFLA 02-290 accumulated similar TDW as in the
of N supply. HN concentration when they were co-inoculated with
Considering the TDW accumulated by the plants, simi- CIAT 899. Unfolding the inoculation factor in each form
lar results were found for UFLA 02-293, UFLA 03-107, of N supply, the treatments with UFPI B3-9, UFLA
UFLA 04-227, and UFLA 04-885, which did not differ 02-281, UFLA 02-286, UFLA 02-290, UFLA 02-293,
among the forms of N supply, and for the other treatments UFLA 04-195, and UFLA 04-227 exhibited greater plant
that developed more in HN (Table 3). Plants inoculated growth than inoculation with CIAT 899 alone. Among the

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Archives of Microbiology (2023) 205:209 Page 7 of 14  209

Fig. 1  Principal component
analysis summarizing the results
of the biocontrol experiment.
Scores of the treatments means
are displayed. Diameter size
indicates plant growth in terms
of total dry weight (TDW). PH,
GERM, RDW, SDW, TDW,
DI, and AUDPC stand for plant
height, germination index, root
dry weight, shoot dry weight,
total dry weight, disease index,
and area under the disease pro-
gress curve, respectively. The
types of inoculation stand for
the inoculation, co-inoculation
(with CIAT 899 strain), or non-
inoculated (control) with the
studied strains

Fig. 2  Effect on disease index (DI) of lima bean co-inoculated with 150, and 200 mg ­kg−1. Higher values indicate lower plant tolerance to
PGPR strains and Rhizobium tropici CIAT 899 in substrate infected damping-off disease
with progressive Rhizoctonia solani inoculum doses of 0, 50, 100,

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Page 8 of 14 Archives of Microbiology (2023) 205:209

Table 3  Total dry weight (TDW), shoot dry weight (SDW), root dry weight (RDW), and shoot nitrogen accumulation (SNA) of lima bean plants
inoculated with PGPR under different forms of N supply
Treatment TDW SDW
g ­jar−1
CIAT LN HN CIAT LN HN

UFPI B3-9 4.51 aB 3.14 bB 7.18 bA 3.37 aB 1.88 bC 4.88 cA

UFPI B4-9 3.14 bB 3.18 bB 7.56 bA 1.94 bB 1.94 bB 5.35 bA
UFLA 02-281 3.74 aB 3.03 bB 6.58 bA 2.34 bB 1.91 bB 4.39 cA
UFLA 02-286 4.27 aB 4.77 aB 6.30 bA 3.24 aB 3.73 aB 4.86 cA
UFLA 02-290 3.61 aA 2.33 bB 4.26 cA 2.15 bA 1.55 bA 1.94 dA
UFLA 02-293 4.57 aA 4.70 aA 5.85 bA 2.95 aA 3.52 aA 4.04 cA
UFLA 03-10 1.64 bB 2.24 bB 5.11 cA 0.85 bB 1.54 bB 3.65 cA
UFLA 03-107 2.91 bA 2.26 bA 3.80 cA 1.77 bA 1.42 bA 1.95 dA
UFLA 03-18 3.38 bB 4.57 aB 9.52 aA 1.99 bC 3.41 aB 6.27 aA
UFLA 03-26 2.54 bB 3.57 bB 7.34 bA 1.55 bB 2.25 bB 5.26 bA
UFLA 04-122 3.04 bB 3.09 bB 7.18 bA 1.67 bB 1.98 bB 5.06 bA
UFLA 04-195 3.91 aB 4.74 aB 6.75 bA 2.98 aB 3.37 aB 4.64 cA
UFLA 04-227 3.78 aA 4.20 aA 6.52 bA 2.59 aB 3.28 aB 4.68 cA
UFLA 04-885 2.63 bA 3.34 bA 3.70 cA 1.55 bA 2.16 bA 1.64 dA
Control 3.12 bB 2.68 bB 6.27 bA 1.78 bB 1.29 bB 4.64 cA
Treatment RDW SNA
g ­jar−1 mg ­jar−1
CIAT LN HN CIAT LN HN

UFPI B3-9 1.13 aB 1.26 aB 2.30 aA 93.12 aB 87.01 aB 162.59 bA

UFPI B4-9 1.19 aB 1.25 aB 2.21 aA 67.62 aB 63.35 aB 200.09 aA
UFLA 02-281 1.40 aB 1.12 aB 2.18 aA 29.34 bB 54.46 bB 96.46 cA
UFLA 02-286 1.03 aA 1.06 aA 1.44 aA 60.48 bA 34.50 bA 81.66 dA
UFLA 02-290 1.47 aB 0.78 aB 2.32 aA 73.57 aA 56.90 aA 94.34 cA
UFLA 02-293 1.62 aA 1.18 aA 1.81 aA 80.58 aA 36.59 bB 110.61 cA
UFLA 03-10 0.79 aA 0.70 aA 1.46 aA 15.57 bB 38.75 bB 127.90 cA
UFLA 03-107 1.13 aB 0.84 aB 1.86 aA 59.77 bA 34.09 bA 60.20 dA
UFLA 03-18 1.39 aB 1.16 aB 3.25 aA 105.54 aB 58.15 aB 230.27 aA
UFLA 03-26 0.99 aB 1.32 aB 2.09 aA 56.62 bB 69.69 aB 180.86 bA
UFLA 04-122 1.37 aB 1.11 aB 2.11 aA 45.82 bB 68.72 aB 147.00 bA
UFLA 04-195 0.94 aB 1.37 aB 2.11 aA 74.00 aB 77.61 aB 180.48 bA
UFLA 04-227 1.20 aA 0.93 aA 1.83 aA 79.94 aB 68.49 aB 176.59 bA
UFLA 04-885 1.08 aB 1.18 aB 2.07 aA 46.27 bA 31.50 bA 59.81 dA
Control 1.33 aA 1.39 aA 1.63 aA 48.21 bB 43.85 bB 147.50 bA

CIAT inoculation with Rhizobium tropici CIAT 899 at low mineral N concentration (5.25 mg ­L−1 N), LN low mineral N concentration (5.25 mg
­L−1 N), HN high mineral N concentration (52.5 mg L
­ −1 N)
Means followed by lowercase letters in the same column compare the inoculation factors of a single N supply by the Scott–Knott test (P < 0.05).
Means followed by uppercase letters in the same row compare the N supply factors of a single inoculation by the Scott–Knott test (P < 0.05)

plants fertilized with HN, only inoculation with UFLA 03-107, and UFLA 04-885 (Table 3). Interestingly, plants
03-18 increased TDW. Notably, some inoculations (UFLA inoculated with UFLA 02-293 had similar SNA when co-
02-290, UFLA 03-10, UFLA 03-107, and UFLA 04-885) inoculated with CIAT 899 as when N was supplied at the
reduced plant growth in HN compared to the non-inocu- HN concentration. Within the treatments of co-inoculation
lated control. with CIAT 899 and LN concentration, the PGPR strains
Shoot N accumulation (SNA) was similar among the varied in their response regarding SNA, and some strains
forms of N supply for UFLA 02-286, UFLA 02-290, UFLA accumulated more N than the respective controls. In the HN

13
Archives of Microbiology (2023) 205:209 Page 9 of 14  209

concentration, however, only inoculations with UFPI B4-9 plants with strain CIAT 899 of Rhizobium tropici was able
and UFLA 03-18 were more effective than the non-inocu- to reduce the incidence of the disease on the plants; however,
lated control; in some cases, plants inoculated with some co-inoculation of lima bean with other PGPR of different
strains even accumulated less N than the control. genera along with CIAT 899 was more efficient in control-
The PCA (Fig. 3) shows that almost all treatments that ling the disease and promoting plant growth in soil infected
received a high dose of mineral N clustered and were posi- with the pathogen. The same effect was observed in com-
tively correlated with SNA, SDW, and TDW. Exceptions are mon bean plants co-inoculated with CIAT 899, a strain that
for the strains UFLA 02-290, UFLA 04-885, UFLA 03-107. nodulates common bean, and different PGPR genera (Fer-
Co-inoculated treatments and treatments that received the reira et al. 2020). Rhizobium strains can act as a biocontrol
low N dose grouped separately from those that received the of pathogenic fungi (Buonassisi et al. 1986; Volpiano et al.
high N dose. Presumably, the plant growth variables were 2018). Since CIAT 899 is a strain well adapted to weathered
correlated with nitrogen accumulation, but correlations with tropical soils and is able to provide N through biological
RDW were low or null. N fixation in nodulating species, such as common bean, it
could be an important asset for biological control of Rhizoc-
tonia solani.
Discussion Among the PGPR strains tested, the treatments that
exhibited lower DI also resulted in the highest accumula-
This study provided evidence of the ability of PGPR to con- tion of biomass and a low AUDPC. This is particularly nota-
trol rhizoctoniosis damping-off disease and to improve lima ble for the co-inoculation treatments UFLA 02-281 + CIAT
bean growth under different nitrogen sources. In general, 899, UFLA 02-286 + CIAT 899, UFLA 04-195 + CIAT
PGPR act as a biological control by competing with unfa- 899, UFLA 04-227 + CIAT 899, and UFLA 04-885 + CIAT
vorable microorganisms or producing different compounds 899. The literature reports that co-inoculation of PGPR
that inhibit or eliminate them. Inoculation of lima bean with rhizobia strains may exert more efficient control on

Fig. 3  Principal component
analysis summarizing the results
of the growth promotion experi-
ment. Scores of the treatment
means are displayed. Diameter
size indicates plant growth
in terms of total dry weight
(TDW). RDW, SDW, TDW, and
SNA stand for root dry weight,
shoot dry weight, total dry
weight, and shoot N accumula-
tion, respectively. The colors of
the circles indicate the source
of N for the plants: CIAT stands
for (co)inoculation with CIAT
899 strain; LN received 5.25 mg
­L−1 of N; and HN received
52.5 mg ­L−1 of N

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Page 10 of 14 Archives of Microbiology (2023) 205:209

pathogenic fungi than inoculation with PGPR alone. Our addition to the well-stablished growth promotion traits of
research group confirmed similar results on common bean indole-acetic acid, this hormone can indirectly trigger plant
with these co-inoculation combinations (Ferreira et  al. immune responses, inhibit, or even stimulate fungal develop-
2020). Rhizobium tropici also limited the development of ment (Kulkarni et al. 2013; Fu et al. 2015). Volpiano et al.
R. solani root rot in common bean when co-inoculated with (2018) found a weak, but significant correlation (r = 0.447,
Bacillus subtilis and improved growth and yield (Jensen p = 0.011) between indole-acetic acid produced by Rhizo-
et al. 2002). Co-inoculation of Rhizobium with PGPR from bium spp. and Sclerotium rolfsii growth inhibition, but the
the genera Bacillus and Pseudomonas also helps other legu- suppression of collar rot disease in field trials could also be a
minous plants thrive against pathogenic fungi: chickpea consequence of the growth promotion abilities of Rhizobium
(Hameeda et al. 2010), lentil (Akhtar et al. 2010), and white strains on common bean.
bean (Kalantari et al. 2018). However, there are no reports of The other genera were also reported to produce com-
co-inoculation of lima bean with Rhizobium and other PGPR pounds that promote biocontrol activity. Strains of Pseu-
strains. The effectiveness of biocontrol promoted by different domonas produced siderophore, indole-acetic acid, pro-
PGPR indicates an important approach in bean production teolytic enzymes, such as chitinase, β-1,3-glucanase, and
and may reduce the need for seed chemical treatments. protease, and significantly reduced the disease index of
Lima bean plants were subjected to a progressive increase tomato plants in the presence of R. solani on both glass-
in the dose of the soil pathogen and withstood up to the dose house and field trials (Solanki et al. 2014). Chitinase and
of 100 mg  ­kg−1 when treated with UFLA 04-885 (Pseu- β-1,3-glucanase are directly involved in the degradation of
domonas), UFLA 04-195 (Rhizobium), and UFLA 04-227 fungal cell walls and insect cuticles, thus being considered
(Burkholderia) co-inoculated with CIAT 899 (Rhizobium). important biocontrol enzymes produced by PGPR (Pereira
The PGPR strains used in this experiment in co-inoculation et al. 2007). Species of Burkholderia have been considered
with CIAT 899 also showed significant biocontrol activity as biocontrol agents for a long time. The genus is able to
in the previous experiment, with the lowest disease indices, produce several hydrolytic enzymes such as chitinase and
as well as an increase in plant biomass. Similar results were β-1,3-glucanase and induce systemic resistance (Ahmad
achieved in common bean subjected to increasing soil patho- et al. 2022), as well as to produce secondary metabolites
gen doses (Ferreira et al. 2020). with biocontrol activity, such as pyrrolnitrin, cepacin, and
There are different mechanisms that PGPR use to act as a burkholdin (Biessy et al. 2022). Bacillus, Brevibacillus, and
biocontrol. Direct mechanisms are related to suppression of Paenibacillus spp. are also capable of producing hydrolytic
pathogenic microorganisms through production of antibiot- enzymes and antibiotics (Budi et al. 2000; Raza et al. 2008;
ics, bacteriocins, cyanhydric acid, metabolites, toxins, and Jamali et al. 2020), and secondary metabolites (Arguelles-
enzymes (Raaijmakers et al. 2002; Compant et al. 2005; Jiao Arias et al. 2009; Canova et al. 2010; Jiang et al. 2015) to
et al. 2021). Siderophores are considered another effective control Rhizoctonia solani and other soil pathogens.
mechanism for microorganism biocontrol. The rationale is The genera used in this study, i.e., Bacillus, Brevibacillus
that the extracellular siderophore chelates iron and prevents Burkholderia, Paenibacillus, Pseudomonas, and Rhizobium,
the pathogen from acquiring that essential nutrient (Radziki are often described as biocontrol agents against several plant
et al. 2013). Antibiotics, bacteriocins, and siderophores are pathogens (O’Sullivan and O’Gara 1992; Depoorter et al.
considered the most effective mechanisms for identifying 2016; Rybakova et al. 2016; Das et al. 2017; Miljaković et al.
potential biocontrol strains (Kloepper et al. 1980). Other 2020). These genera are also well known as plant growth pro-
indirect mechanisms also help control pathogens: rhizos- moters and are already recommended worldwide for diverse
phere colonization (Chiarini et al. 1998), induction of sys- growth promotion traits in different plant species (Bhattachar-
temic resistance (Raza et al. 2016) and acquired resistance yya and Jha 2012). Furthermore, to the best of our knowledge,
(Gao et al. 2015), and hormone interaction, such as auxins research on growth promotion and biocontrol in lima beans is
and gibberellins (Kazan and Manners 2009). scarce, and there is a need to verify positive effects of PGPR
Rhizobium strains are able to produce inhibitory com- on this plant species since Phaseolus lunatus is the second
pounds or promote plant resistance through several ways. most important species in the Phaseolus genus (Fofana et al.
CIAT 899 strain was reported to produce volatile com- 1999).
pounds and siderophore to control the pathogenic fungi Plant growth promotion by the strains varied in each form
Sclerotium rolfsii, while other Rhizobium strains produced of N supply. Overall, inoculations with UFPI B3-9 (Paeniba-
considerable amounts of indole-acetic acid (Volpiano et al. cillus sp.), UFLA 02-286 (Brevibacillus sp.), UFLA 02-290
2018). Innumerous volatile compounds affect mycelial (Bacillus megaterium), UFLA 02-293 (Pseudomonas putida),
growth and virulence enzymes such as laccase, a viru- UFLA 04-195 (Rhizobium miluonense), and UFLA 04-227
lence factor that protects the pathogen from plant defense (Burkholderia fungorum) combined with CIAT 899 (R. trop-
molecules (Mayer and Staples 2002; Wheatley 2002). In ici) led to an increase in plant biomass and N accumulation

13
Archives of Microbiology (2023) 205:209 Page 11 of 14  209

in comparison with plants inoculated with CIAT 899 alone. of some PGPR strains on lima bean, and further research
In addition to biocontrol, other plant growth promotion traits is necessary to identify the mechanisms of biocontrol of
from strains of these genera have been reported, and positive these strains as well as their effects under field conditions.
results on growth, nodulation, and N fixation in leguminous We believe that these outcomes are important first steps to
plants have been found from co-inoculation of Rhizobium with improve lima bean production either by promoting plant
Bacillus (Rajendran et al. 2008, Korir et al. 2017), Brevibacil- growth or by managing R. solani damping-off, especially
lus (Abbas et al. 2018), Burkholderia (Oliveira-Longatti et al. considering the low cost of inoculation for a crop mostly
2013), Paenibacillus (Korir et al. 2017), and Pseudomonas produced by small farmers.
(Tilak et al. 2006).
Acknowledgements  We thank the Conselho Nacional de Desenvolvi-
In some cases, the combinations of PGPR strains with mento Científico e Tecnológico (CNPq), the Coordenação de Aper-
CIAT 899 were able to increase plant biomass and accumu- feiçoamento de Pessoal de Nível Superior (Capes), and the Fundação
lated N as much as in the treatments that received mineral de Amparo à Pesquisa do Estado de Minas Gerais (Fapemig) for fund-
nitrogen. The co-inoculations UFLA 02-290 + CIAT 899 ing and granting scholarships; we also thank the CNPq for scientific
productivity scholarships granted to FHVM (No. 317266/2021-7) and
(Bacillus megaterium + R. tropici), UFPI B3-9 + CIAT 889 FMSM (No. 310015/2021-9). This research is associated with the Insti-
(Paenibacillus sp. + R. tropici), and UFLA 02-293 + CIAT tuto Nacional de Ciência e Tecnologia (National Institute of Science
899 (Pseudomonas putida + R. tropici) were able to achieve and Technology—Soil Biodiversity/ INCT-CNPq).
values of total dry weight, shoot dry weight, and accumulated
Author contributions  Conceptualization: LVM Ferreira, FHV Medei-
N, respectively, equal to application of the highest mineral ros, FMS Moreira; Methodology: LVM Ferreira, FHV Medeiros,
N concentration and higher than application of the low min- FMS Moreira; Formal analysis and investigation: LVM Ferreira, F
eral N concentration. Nodulation, however, was not found Carvalho, JFC Andrade, RA Leite; Writing - original draft: LVM Fer-
in this study. Lima bean is able to nodulate especially with reira, RA Leite, FMS Moreira; Funding acquisition: FHV Medeiros,
FMS Moreira; Resources: FHV Medeiros, FMS Moreira; Supervision:
Bradyrhizobium (Ormeño-Orrillo et al. 2006), as well as effi- FMS Moreira;
ciently fix ­N2 through symbiosis with this genus (Costa et al.
2017). Since nodulation with Rhizobium is not possible, the Funding  Coordenação de Aperfeiçoamento de Pessoal de Nível
growth promotion by the Rhizobium strains were other than Superior,Conselho Nacional de Desenvolvimento Científico e
Tecnológico,Fundação de Amparo à Pesquisa do Estado de Minas
symbiotic ­N2 fixation, and the positive effect was enhanced Gerais.
by co-inoculation with other PGPR genera. Nevertheless, the
combination of CIAT 899 and PGPR strains from other genera Data availability  The datasets generated during and/or analysed dur-
enabled more plant N acquisition despite the lack of nodula- ing the current study are available from the corresponding author on
reasonable request.
tion, indicating a favorable synergistic effect of these strains,
with positive consequences for lima bean growth and an asym- Declarations 
biotic ­N2 fixation effect.
Conflicts of interest  The authors have no conflicts of interest to de-
clare. 
Conclusions

Most strains were able to manage damping-off and pro-

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