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Applied Soil Ecology

journal homepage: www.elsevier.com/locate/apsoil

Scaling from the growth chamber to the greenhouse to the field:Demonstration of diminishing effects of mitigation of salinity in peppersinoculated with plant growth-promoting bacterium and humic acids

Macario Bacilioa, Manuel Morenoa, D. Raul Lopez-Aguilarb, Yoav Bashana,c,d,⁎

a Environmental Microbiology Group, Northwestern Center for Biological Research (CIBNOR), Av. IPN 195, La Paz, B.C.S. 23096, Mexicob Programa de Agricultura de Zonas Áridas, Guerrero Negro unit, Northwestern Center for Biological Research (CIBNOR), Av. IPN 195, La Paz, B.C.S. 23096, Mexicoc The Bashan Institute of Science, 1730 Post Oak Court, Auburn AL 36830, USAd Dept of Entomology and Plant Pathology, 301 Funchess Hall, Auburn University, Auburn, AL 36849, USA

A R T I C L E I N F O

This study is dedicated for the memory of therhizophere researcher Dr. Michael Schmid (Big-Mike; 1968–2016) of Helmholz-Zentrum,Munich, Germany.

Keywords:Humic acidsInoculationPepperPlant growth-promoting bacteriaSalinity

A B S T R A C T

The capacity of pepper plants to alleviate salt stress when inoculated with the plant growth-promoting bacteria(PGPB) Pseudomonas stuzeri and/or supplementation with humic acids was compared under in vitro conditions ina growth chamber (three independent experiments), greenhouse (four experiments) and field conditions (twoexperiments). Although inoculation with PGPB or humic acids significantly mitigated negative effects of salinityon germinating pepper seedlings under in vitro conditions, the effect was far less marked under greenhouseconditions and almost non-existent under field conditions, having no impact on yield of peppers under salineconditions. This study demonstrates that the improvement in growth with PGPB and humic acids in vitromay notbe scalable from the laboratory to greenhouse and to field conditions. Not all PGPB improvements scale from thelaboratory, greenhouse to field conditions and that the potential that laboratory results may not scale is a factorto be considered in this research field.

1. Introduction

When carrying out experiments with biotic agents with plants, acommon assumption made is that experimental results or effects ofbiotic agents with plants will scale, that test tests under laboratoryconditions will be similar to those in the greenhouse and under fieldcondition. In agro-practice, it is not uncommon to follow a procedure ofscaling-up initial experiments done in the laboratory to the field. Thiscommon ideal assumption is that experiments in the laboratory willscale linearly and indicate the best treatments to be implemented laterat a large scale by the appropriate agro-industry.

While sometimes experimental conditions carried out in the la-boratory are scalable and correlate well with greenhouse experiments(Bashan et al., 2009; Beattie et al., 1989; Brown et al., 1999; Kumar andPoehling, 2006; Owusu-Bennoah et al., 2002; Vleeshouwers et al.,1999; Willits and Peet, 2001), other times this is not the case. It is notuncommon at times, to find no positive correlation of an effect betweenthe same applied experimental conditions under laboratory, greenhouseand/or field conditions (Dorrance and Inglis, 1997; Foolad et al., 2000),other times some effects are not conclusive (Kim et al., 2000) or evennegative effects are observed (Antoun et al., 1998; Kesselmeier, 2001).

When plant growth-promoting (rhizo)bacteria (PGPB/PGPR) are ap-plied to increase plant performance, every major review on the topicstates that PGPB/PGPR can stimulate plant growth under laboratoryconditions, but when they are applied at a field-scale they fail in manycases to have the predicted impact (Bashan and de-Bashan, 2010;Bashan et al., 2014; Calvo et al., 2014; Glick, 1995; Glick et al., 2007;Martínez-Viveros et al., 2010).

Although most literature on effects of PGPB/PGPR on plant growthshows significant improvements in plant performances and yield(Bashan et al., 2014; Calvo et al., 2014; de-Bashan et al., 2012), thereality is that failures to achieve the results desired are frequent and areseldom reported, especially by businesses promoting PGPB-based in-oculation technology. In an older review, it was calculated that only70% of inoculation of various cereals with the PGPB Azospirillum sp.were effective (Okon and Labandera-Gonzalez, 1994), where the vastmajority of the literature shows positive results.

Several of our inoculation experiments over the last two decadesfailed to yield the predicted positive plant response using various PGPBstrains that were presumed to be potentially highly beneficial to plantswhen laboratory results were scaled up to the field. These results,therefore, were never published (unpublished data). As a consequence,

http://dx.doi.org/10.1016/j.apsoil.2017.07.002Received 8 November 2016; Received in revised form 14 June 2017; Accepted 2 July 2017

⁎ Corresponding author at: The Bashan Institute of Science, 1730 Post Oak Court, Auburn, AL 36830, USA.E-mail addresses: bashan@bashanfoundation.org, ybashan@cibnor.mx (Y. Bashan).

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our current hypothesis was that in some combinations of PGPB-plantthere is no correlation between successful results obtained in vitro andperformance of the same combination under greenhouse and fieldconditions. To test this hypothesis we used an experimental modelemploying the diazotroph PGPB Pseudomonas stuzeri that showed sev-eral positive responses on the growth of pepper plants under green-house conditions in combination with humic acids (Bacilio et al., 2016).This comparative study compared the growth of peppers under similarexperimental conditions in vitro and in two field experiments. A generalanalysis was done comparing all these experiments with experimentaldata published before of very similar experiments done under green-house condition (Bacilio et al., 2016). The comparative experimentalsystem of mitigation of salinity in peppers with humic acids and PGPBfrom in vitro studies to the field served solely as an experimental modelfor this comparison. Understanding the specific reasons of mitigation ofsalinity in peppers was not the focus of this investigation.

2. Materials and methods

2.1. Plants

Bell pepper (Capsicum annuum L.) cv. Jupiter (Syngenta Seeds,Boise, Idaho) and cv. Ancho San Luis (Crown Seeds, California) wereused. The first cultivar is relatively tolerant to salinity and the secondcultivar is relatively susceptible (Bacilio et al., 2016). Seeds were firsttreated with 2% Tween-20 (#P2287, Sigma, St. Luis, Mo) washed indistilled water five times, then disinfected in 3% commercial NaOCl(Clotalex, Mexico City, Mexico) for 5 min, and rinsed several times insterile tap water. This treatment yield germination at a level of 100%within 6 days for cv. Jupiter and within 10 days for cv. Ancho San Luis.

2.2. PGPB, growth conditions and inoculant preparation

Inoculant preparation and inoculation of plants followed establishedguidelines (Bashan et al., 2016). The desert diazotroph PGPB Pseudo-monas stutzeri strain TREC (GenBank accession number: JX014305;Puente and Bashan, 1994) was used for all experiments. A single colonywas cultured in 250-mL Erlenmeyer flasks containing 150 mL of nu-trient broth (NB, Fluka) and incubated at 32 ± 4 °C, 120 rpm for 48 h.Bacteria were harvested by centrifugation at 2683 × g for 10 min a4 ± 1 °C. Bacterial cells were washed three times with saline solution(0.85%, w/v, NaCl) to eliminate all residues of nutrient broth. Finally,the bacterial suspension was diluted in the latter saline solution to106 cfu mL−1. This type of suspension served as inoculant for in vitroexperiments. For experiments under field conditions, an initial sus-pension of 109 cfu mL−1 was the source of PGPB for formulating drymicrobead alginate inoculant (100–200 μm) that was produced ac-cording to Bashan et al. (2002). This inoculant had a population of108 cfu g−1 beads.

2.3. In vitro growth chamber screening experiments

Initially all treatments of humic acids and bacterial inoculants werescreened for effectiveness in Petri dish setups. Each treatment containedfour standard size Petri dishes. Each Petri dish contained two layers oftowel paper and moist filter paper (Whatman #1, Sigma-Aldrich)socked with 0, 25, 50 and 75 mM NaCl (in distilled water). Seeds weredisinfected as described above. Seeds were soaked for two hours insterile distilled water. Then, 20 seeds per plate were placed on the filterpaper, and incubated in a growth chamber (Conviron, Winnipeg,Canada) at 28 ± 2 °C and 80% relative humidity for the first 8 days inthe dark and later under 200 μmol photon m−2 s−1. Inoculum con-centration per Petri dish (where applicable) was 106 CFU mL−1 and theconcentration of commercial humic acids (active ingredients: minimum65% humic acids and minimum 85% potassium humate; 93–98%water-soluble; Enersol SC, American Colloid, Arlington Heights, IL) was

1000 mg L−1. After 21 days of incubation, the following parameterswere measured: shoot length, shoot diameter, root length, number ofsecondary roots, length of root hairs and root hair surface. Length ofroot hairs and root hair surface were measured by sampling one cen-timeter segments of roots from the root hair zone, two centimeter aboveroot tips (three segments per treatment). Root segments were placed ina Petri dish, stained with toluidine blue for three minutes at roomtemperature, and rinsed with distilled water. Finally, photomicrographswere taken under a light microscope (Olympus BX-45, Japan) con-nected to an image analyzer (Image ProPlus 4.5, Media Cybernetics,Silver Spring, MD) in each of these root segment chosen by random atsample size of 200 μm2. The image analyzer used the software ImagePro-plus version 4.1, and photographic camera model Cool Snap MediaCybernetics. Length of root hair were measured referred to 200 μ2surface of roots.

2.4. Greenhouse experiments

Four independent greenhouse experiments having identical treat-ments (listed below) were done and described in detail in a previousstudy (Bacilio et al., 2016).

2.5. Field experiments

Two experiments under field conditions were done in the experi-mental field of the Guerrero Negro Experimental Station of CIBNORlocated in the Vizcaíno Desert, Baja California Sur, Mexico at27°57′32″N 114°03′22″W. This sandy desert area bordering the PacificOcean is characterized by having frequent fog events, annual pre-cipitation of ∼80 mm, strong winds and high evaporation rates. Thetemperature during the experiments ranged between 32.8 and 6.3 °C.The soil in the area is classified as Aridosoles (Endo et al., 2000). Thephysicochemical characteristics of the two specific soils used in thisstudy (saline and non-saline soils) were done by a service unit and aredescribed in Table 1 using standard soil testing techniques authorizedby the Mexican government. Soils were collected from the plough layer(upper 40 cm of the soil) from an uncultivated land at CIBNOR –Guerrero Negro Experimental Station. The soil was air-dried, groundedand screened to pass through a 2 mm sieve.

Compost used as a practical source of humic acids in these fieldexperiments. Compost produced from cultivated cruciferous waste(cauliflower and broccoli) and dairy cow manure at a ratio of 1:2(soil:compost, v/v, resembling the recommended level of compost ap-plication in desert soil agriculture in Baja California Sur, Mexico). Thephysicochemical composition and characteristics of the composts were:

Table 1Chemical and physicochemical characteristics of the two soils used in the field experi-ment.

Characteristic Non saline soil Saline soil

Textural class Sand SandBulk density, (g cm−3) 1.51–1.58 1.53–1.57pH 8.21–9.12 8.32–9.38EC, (dS m−1) 1.32–2.17 4.25–4.97ESP (%) 6.2–11.4 23.4–31.9CaCO3 (%) 3.18–5.05 3.82–4.19CEC, cmol(+) kg−1 1.73–4.27 1.68–5.03Organic matter (%) 0.19–0.38 0.14–0.45Total nitrogen (g kg−1) 0.35–0.84 0.28–0.75P-Olsen (mg kg−1) 30.1–44.7 24.7–41.2Sol + Exch Ca2+, (g kg−1) 0.63–1.23 0.73–1.88Sol + Exch Mg2+ (g kg−1) 0.18–0.25 0.28–0.52Sol + Exch K+ (g kg−1) 0.32–0.47 0.49–0.76Sol + Exch Na+ (g kg−1) 0.12–0.38 1.61–1.93Cl− (g kg−1) 0.17–0.43 1.97–3.48

EC = Electrical conductivity; ESP = Exchangeable sodium percentage; CEC = Cationexchange capacity

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(in%) total N, 1.9; nitrate, 1.32; P, 0.8; K, 0.63; Na, 0.27; Fe, 0.31; Mn,0.04; Zn, 0.01; Cl, 0.08; SO4, 0.13; organic matter, 10.9; electricalconductivity (in mS cm−1, 25 °C), 1.04; pH 9.2. Humic acid con-centration was 5.1% (Bacilio et al., 2003). Compost particles weresieved to 2 mm in diameter. Application of compost is more than justsolely the application of humic acids and may introduce some bias intothe results. However, these field experiments were conducted in a si-milar manner to commercial cultivation of peppers in this highly sandysoil, a dune-like soil, and without compost that helps the pepper plantssustain moisture they cannot grow there.

Plants were initially grown in a mixture of peat:silica sand (1:1, V/V). Commercial plastic trays of 128 cavities, 4 × 4 × 4 cm (each plug)(Ningbo Evergreen Irritech, Zhejiang, P.R. China) were used. A singleseed was sown in each planting hole, 1 cm below the surface and thetray was watered with tap water until field capacity. Trays were in-cubated in an uncontrolled, transparent, plastic walk-in tunnel with

average night time temperature of 21 °C and average day time tem-perature of 30 °C. The seeds were irrigated with distilled water untilnearly saturation point every third day.

Pepper seedlings (28 d, salt-resistant cultivar and 34 d, salt-sus-ceptible cultivar) were transplanted to commercial plastic trays(64 × 32 × 12 cm, l/w/d) used for commercial cultivation of peppersin this area. Each tray contained 14 kg of one of the two soils used andtwo seedlings. In treatments containing compost, the percentage ofcompost (10–30%) was added to the soil per dry weight. Inoculationwas done by adding one gram of dry inoculant containing 108 cfu g−1

to each of the planting holes. Plants were immediately watered to fieldcapacity with tap water.

This setup of individual trays placed on the ground has severaladvantages over direct planting of the plant in the soil. It allows forprecise measurement of the effects on plant growth parameters (detailsbelow), avoids dilution of the treatments (PGPB and compost) to nearby

Fig. 1. Effect of (1) application of humic acids, (2) inoculation with the PGPB Pseudomonas stutzeri, and (3) combination of application of humid acids and inoculation with the PGPB onplant parameters in a salt-susceptible cultivar of pepper (a,b,c) and salt-resistant pepper (d,e,f) subjected to four concentration of salinity (0–75 mM) in vitro. In each subfigure, separately,columns followed by different letters differ significantly by one-way ANOVA and then by Fisher LSD post-hoc analysis at P < 0.05. Unlabeled columns are not significantly different.Whiskers represent standard error (SE). Missing whiskers indicate minimal SE.

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soil and avoids loss of root mass at harvest time. All experimental trayswere placed in the same field were soil samples were obtained andremained there throughout the experiment to provide the same en-vironmental conditions, drip irrigation and drip nutrition as commer-cial plants growing directly in the soil. The plants were initially dripirrigated daily for 14 days with 0.5 L each with nutrient solutioncomposed of: 14 mM nitrate, 1.0 mM ammonium, 0.5 mM P, 3.5 mM K,4.5 mM Ca, 1.5 mM Mg, 1.5 mM S, 25 μM Fe, 10 μMMn, 2.0 μM Zn and

0.3 μM Cu. Later, the level of nutrition provided was increased withplant development and tap water was added routinely and auto-matically by drip irrigation system according to the environmentalconditions to maintain the soil at a constant water field capacitythroughout the growing season similar to nearby commercial cultiva-tion. Because no diseases or insects affected the plants, no pesticideswere used.

Fig. 2. Effect of (1) application of compost (containing humic acids) at 3 concentrations, (2) inoculation with the PGPB Pseudomonas stutzeri, and (3) combination of application ofcompost and inoculation with the PGPB on plant parameters in salt susceptible and salt-resistant cultivars of pepper growing in the field in non-saline soil. In each subfigure, columnsfollowed by different letter differ significantly by one-way ANOVA and then by Fisher LSD post-hoc analysis at P < 0.05. Unlabeled columns are not significantly different. Whiskersrepresent standard error.

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2.5.1. Plant sampling, harvesting and analysesPlants were harvested at 110 days post treatments after transferring

each of the trays from the field to the nearby laboratory at CIBNOR-Guerrero Negro experimental station. There, plants were separated intoroots, stem, leaves and fruits. The roots extracted from trays were first

dipped in a bucket filled with tap water and gradually washed until allsoil particles were removed. Then roots were washed with distilledwater for 3 min and excess water was blotted by paper towel. Foliararea was measured by an area integrator (ACC-400, Hayashi-Denko,Osaka, Japan) All vegetative plant parts (apart from fruits) were placed

Fig. 2. (continued)

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in paper bags and dried at 65 °C in an oven (Yamato DK-85, California)for three days until constant weight to determine dry weight. Height ofplants was measured from the base of the stem to the apical meristem.Diameter of the stem was measured by digital caliper, 2 cm above thesoil surface. Fruit yield was measured by digital balance with precisionof 0.0001 g. Chlorophyll content was measured at the same hour (11AM) by a portable chlorophyll meter Minolta SPAD-502 (KonicaMinolta, Osaka, Japan). The readings were done on four fully expended

leaves (two readings per leaf, on leaves 3–6 counting from the base ofthe plant). The eight readings were registered as the SPAD (Soil PlantAnalysis Development) chlorophyll value of the plant (Ling et al., 2011;Uddling et al., 2007).

2.6. Experimental design and statistical analysis

The in vitro and greenhouse experiments used the same eight

Fig. 3. Effect of (1) application of compost (containing humic acids) at 3 concentrations, (2) inoculation with the PGPB Pseudomonas stutzeri, and (3) combination of application ofcompost and inoculation with the PGPB on plant parameters in salt susceptible and salt-resistant cultivars of pepper growing in the field in saline soil. In each subfigure, columns followedby different letter differ significantly by one-way ANOVA and then by Fisher LSD post-hoc analysis at P < 0.05. Unlabeled columns are not significantly different. Whiskers representstandard error.

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treatments described below. Apart from the inherent difference becauseof the type of environment (e.g. in vitro experiments did not containsoil, while greenhouse experiments did) the other variables are com-parable for the purpose of this study. The treatments in vitro and in thegreenhouse experiments were: Inoculation with the PGPB, applicationof humic acids; inoculation with PGPB + application of humic acids

and non inoculated controls, all under 4 salinity levels: 0, 25, 50,75 mM NaCl.

The treatments in the field were very similar and involved two typesof soil, saline and non- saline soil and were: 1) soil without any treat-ment; 2) soil with 10% compost; 3) soil with 20% compost; 4) soil with30% compost; 5) soil inoculated with PGPB; 6) soil with 10% compost

Fig. 3. (continued)

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+ inoculation with inoculation with PGPB; 7) soil with 20% compost+ inoculation with PGPB; 8) soil with 30% compost + inoculationwith PGPB. The compost contained 5.1% humic acids. Therefore 10%compost corresponds to the application of 5600 mg of humic acids kgsoil−1, 20% compost provided 11,330 mg humic acids·kg soil−1 and30% compost provided 17,000 mg of humic acids kg soil−1.

In vitro experiments were conducted with four replicates where onePetri dish containing 20 seedlings served as a replicate. Each experi-ment was repeated three times. Each treatment in the field experimentshad 5 replicates where one tray with two plants served as a replicate.These experiments were repeated twice.

The two repetitions of the greenhouse experiments were identical.Each experiment had 5 or 6 replicates per treatment where one re-plicate was a pot containing one plant. Because data from each re-petition of the experiments (greenhouse and laboratory, separately)were very similar, data were combined and analyzed together (Bacilioet al., 2016).

Data for all types of experiments were first combined from the re-peated individual experiments and analyzed first by ANOVA and thenby Fisher LSD post-hoc analysis or Student’s t-test at P < 0.05. Allstatistical analyses were done using Statistica v. 6.0 (Statsoft, TolsaOK).

3. Results

3.1. Effect of inoculation with PGPB and application of humic acids aloneand combined on development of salt-resistant and salt-susceptible peppersseedlings in vitro and under greenhouse conditions

Seedling development after 21 days of incubation under increasingsalt concentration (0–75 mM) was tested for shoot length and diameterand length of roots. In the salt susceptible cultivar, some of the treat-ments showed positive improvement mostly under salt concentration of50 mM or less (Figs. 1 a,b,c and 5a). Similar phenomena occurred in thesalt-resistant cultivar albeit to a lower extent (Figs. 1d,e,f, 5a).

Under greenhouse condition the salt resistant cultivar respondedmore positively to the treatments than the salt susceptible cultivar(Fig. 5b, detailed results in Bacilio et al., 2016).

3.2. Effect of inoculation with PGPB and application of compost alone andcombined on development of salt-resistant and salt-susceptible peppersseedlings under field conditions

Field experiments were done in the format of commercial cultiva-tion of peppers in the region. In the non-saline soil, no major effects

Fig. 4. Effect of (1) application of compost (containing humic acids) at 3 concentrations, (2) inoculation with the PGPB Pseudomonas stutzeri, and (3) combination of application ofcompost and inoculation with the PGPB on yield of pepper plant in salt susceptible and salt-resistant cultivars of pepper growing in the field in non-saline and saline soils. In eachsubfigure, columns followed by different letter differ significantly by one-way ANOVA and then by Fisher LSD post-hoc analysis at P < 0.05. Unlabeled columns are not significantlydifferent. Whiskers represent standard error.

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were found in plant growth parameters of the salt-susceptible cultivarwith any of the treatments except for the treatment with 30% compost.This treatment significantly affected plant height (Fig. 2a) and foliararea (Fig. 2e). Field yield increased where 20% and 30% compost wereadded but in no other treatment (Fig. 4a). The same phenomena wereobserved in salt-resistant cultivar where only the stem dry weight waspositively affected by amendment of 10% and 30% compost (Fig. 2n)and no effect on yield was recorded (Fig. 4b)

In salt-affected soil the salt-susceptible cultivar showed a similareffect with the compost application in recorded foliar area (30% com-post and 30% compost plus PGPB, Fig. 3e) and stem diameter (20% and30% compost and 30% compost plus PGPB; Fig. 3k). No effect on theyield was detected (Fig. 4c). In the salt-resistant cultivar several plantparameters were significantly enhanced such as height (PGPB, Fig. 3b),number of leaves (30% compost plus PGPB, Fig. 3d), foliar area (10%compost plus PGPB, Fig. 3f), dry weight of leaves by several treatments(Fig. 3h), stem diameter (PGPB and 10% compost plus PGPB, Fig. 3l),stem dry weight (10% compost and PGPB treatment, Fig. 3n). None ofthese positive responses enhanced pepper yield in saline soil (Fig. 4d).

3.3. General analysis of in vitro vs. greenhouse vs. field experiments

Analyzing entire datasets of in vitro experiments, field experiments(this study) and greenhouse experiments published before by Bacilioet al. (2016) revealed that the treatments of PGPB and humic acids havesome positive merits in improving growth of the pepper plant undersaline soil conditions but did not show clear-cut unequivocal effects.Some plant parameters were positively affected at some saline con-centrations and some were not. Although two types of peppers wereused, salt-resistant cultivar and salt-susceptible cultivars, the effectdepended on the type of test rather than the inherent salt tolerance ofthe plants. Salt susceptible seedlings reacted more positively under invitro conditions whereas salt-resistant cultivar reacted more positivelyunder greenhouse and field conditions (Fig. 5). Yet, no effects resultedin enhancing pepper yield in the field in saline soil.

The most notable phenomenon was the diminishing effect from in

vitro studies to field experiments. As the experiments progressed ac-cording to the traditional paradigm from the laboratory tests to field-scale cultivation the beneficial effects diminished, and there was noimprovement or increase in fruit yield (Fig. 5, compare positive re-sponses in each cultivation scheme).

4. Discussion

The literature search that was carried out for this study in scientificdatabases showed that the majority of formal publications regardinginteractions of PGPB/PGPR and plants describe either in vitro (growthchamber) or greenhouse growth conditions, sometimes both in thesame publication with only a few publications containing field data.These publications almost exclusively present successes, big and small.Like most negative results in science, failure in achieving growth pro-motion by PGPB/PGPR is rarely published. When failure is published, itis usually associated with a success in another aspect of the interactionsuch as vigorous initial growth ending with low final yield. In vitro testsare commonly used for screenings of PGPB/PGPR (Bashan et al., 2016),and of these only a few of the treatments are tested in a greenhouse, andeven fewer in the field. Comparisons among performances of the sametreatments under similar laboratory-greenhouse-field studies are rare inplant science (for reference see Introduction) and even less in PGPB/PGPR (Kumar et al., 2016) and none were found using pepper plants asfar as the authors are aware.

The relationship between effects on the PGPB cell or on plantgrowth promotion across various growth conditions is commonplace inPGPB-inoculated plants. In Azospirillum sp., a very common PGPB, arelationship was found between characteristics of the bacterium mea-sured in vitro and the same features measured under greenhouse or fieldcondition. For example, A. brasilense has a significant motility in agarand solutions in vitro both horizontally and vertically (Barak et al.,1983; Bashan and Holguin, 1994; Bashan and Levanony, 1989;Reinhold et al., 1985) and a similar capacity was measured in the soil inthe laboratory (Bashan 1986a; Bashan and Levanony, 1987) and fieldcondition (Bashan and Holguin, 1995). The same effect has been

Fig. 5. General analysis of the effect of (1) application of humic acids, (2) inoculation with the PGPB Pseudomonas stutzeri, and (3) combination of application of humid acids andinoculation with PGPB on plant parameters in a salt-tolerant pepper and a salt-susceptible pepper subjected to different concentration of salt. a) salt-susceptible and salt-resistant cultivarunder in vitro conditions; b) salt-susceptible and salt-resistant cultivar-under greenhouse conditions (Bacilio et al., 2016); c) salt-susceptible and salt-resistant cultivar-under fieldconditions.

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observed with plants, although not by direct comparison, as done in thisstudy, but rather in similar studies carried out by other independentresearchers. For example: significant enhancement of growth and yieldwas found in the laboratory (Carrillo et al., 2002; Cassán et al., 2009;Kapulnik et al., 1985; Lin et al., 1983; Zimmer et al., 1988) greenhouse(Bashan, 1986b; Bashan and de-Bashan, 2002; Bashan and Gonzalez,1999; Isawa et al., 2010; Mehnaz and Lazarovits, 2006) and fieldconditions (Kapulnik et al., 1983, 1987; Millet and Feldman, 1984;Millet et al., 1985) culminating in high percentage of success undercommercial cultivation (Dıaz-Zorita and Fernandez-Canigia, 2009;Hungria et al., 2010). Similar phenomena occurred with inoculation ofvarious species of the PGPB Bacillus sp., for example: 1) Bacillus amy-loliquefaciens in the laboratory (Carvalhais et al., 2013; Dietel et al.,2013) greenhouse (Chowdhury et al., 2013) and field conditions(Borriss, 2011; Yao et al., 2006). 2) Bacillus subtilis in the laboratory(Kumar et al., 2011a, b) greenhouse (Kumar et al., 2011b; Ryu et al.,2007; Zhang et al., 2010) and field conditions (Kokalis-Burelle et al.,2006) and, 3) B. pumilus under laboratory (Zhang et al., 2002); green-house (Zahng et al., 2010; Zehnder et al., 2000) and field condition(Zehnder et al., 2000). Similar results have also been reported forPseudomonas putida in the laboratory (Glick et al., 1994; Lifshitz et al.,1987) greenhouse (Mehnaz and Lazarovits, 2006; Yao et al., 2010) andfield conditions (Gravel et al., 2007; Grimes and Mount, 1984). Con-sidering the wealth of published information, it appears that the as-sumption that scaling from the laboratory to larger scales is possible.Yet, most reviews on PGPB/PGPR inoculation advice that laboratoryexperiments cannot predict well the outcome of inoculation scale-up ofplants to the field. This suggestion is probably based on unpublishednegative results that the authors of the reviews had (see Introductionfor references).

This study showed that one PGPB, P. stuzeri alone or combined withhumic acids, does not follow linear scale-up from the laboratory to thefield. Irrespective of the promising results obtained in the in vitro ex-periments, the performance of this PGPB under standard greenhouseconditions was smaller and the effects were further reduced under fieldconditions and completely disappeared when yield was harvested.These temporary improvements in plant growth under controlled con-ditions can be attributed to the known effect of humic acids on miti-gation of soil salinity (Calvo et al., 2014; Ounia et al., 2014).

The reasons given by other independent studies for the lack ofcorrelation between results obtained from laboratory studies and fieldstudy are mostly speculative and based more on common sense than onpresented data. For example, Antoun et al. (1998) speculated that themost probable cause is over-production of substances that are beneficialto plant growth at low concentration, like IAA or HCN, which arecommonly detected in vitro but rarely in the field. Other studies refer tolaboratory studies employing the same size of leaves, same age of plantand precise inoculation method that are by default more accuratecompared to field studies due to circumstances commonly found underfield conditions. It was also claimed that plant parts under laboratorycondition do not behave similarly as intact plants growing in the field(Dorrance et al., 1997; Kim et al., 2000). Other study indicated thatthese discrepancies may be related to environmental conditions pre-vailing in the field such as climate, water availability and air pollutionand not in the laboratory (Kesselmeier, 2001), where other study waspurely empirical and did not provide any explanation (Foolad et al.,2000). In our set of experiments from in vitro testing to the field, weattributed the large differences in success to extreme and changingenvironments in the desert field vs. controlled conditions in the la-boratory, including possibly different management strategies due to the

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experiments being carried out by a different team.Taking into account the results of the present study and the previous

study (Bacilio et al., 2016) together as evidentiary examples, we pro-pose that this phenomenon is perhaps more widespread in PGPB/PGPRexperimentation, but apparently it is under-reported. This under re-porting of failures was previously proposed for inoculation with thePGPB Azospirillum spp. where a high percentage of field experimentswere successful, but not all (Okon and Labandera-Gonzalez, 1994;Bashan et al., 2004; Dıaz-Zorita and Fernandez-Canigia, 2009). Con-sequently, we believe that there is scientific value in reporting negativeexperimental results in general not only in this microbiological field ofimproving plant yield with PGPB, but in all scientific fields.

Acknowledgments

At CIBNOR campus La Paz and campus Guerrero Negro we thankVicente Verdugo, Mario Benson Rosas, Carmen Rodríguez Jaramillo,Jorge Cobos Anaya, Guillermo García Cortéz, Adan Trejo, Lupita López,Alicia Jiménez, Dennise Covarrubias, Patricia Castillón, GuillermoGonzález, and Juan Pablo Hernández for technical assistance. We thankMichael V. Cordoba for editorial suggestions and English editing. Thisstudy was supported by Consejo Nacional de Ciencia y Tecnologia(CONACYT-Basic Science-2004, contract 43069 to MB) of Mexico andtime for writing by The Bashan Foundation, USA. This is contribution2017-021 of the Bashan Institute of Science, USA.

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