Symbiotic Nitrogen Fixation in Non-legumes: N-supply, Yield and Quality Effects in Tomato and Pepper

Progress report for GNE18-186

Project Type: Graduate Student
Funds awarded in 2018: $14,850.00
Projected End Date: 02/28/2021
Grant Recipient: The Pennsylvania State University
Region: Northeast
State: Pennsylvania
Graduate Student:
Faculty Advisor:
Dr. Sjoerd Duiker
Penn State University
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Project Information

Project Objectives:

Objectives: The goal of this research is to evaluate the effect of inoculation of non-legumes with the BNF bacterium, Gluconacetobater diazotrophicus (Gd) . My specific objectives are:

1. Detail the impact of Gd inoculation of tomatoes and peppers on vegetative plant growth and fruit yield.

2. Evaluate if BNF with Gd can replace external N requirements

3. Assess effects of Gd on fruit nutrient content

4. Evaluate the economic value of Gd inoculation

5. Use mass balancing techniques to evaluate the efficacy and amount of atmospheric nitrogen capture in inoculated plants.

Introduction:

With an increasing population placing increased demands on agricultural output, new techniques to improve crop production and nutrition are more of a necessity than ever. Coupling this with continued pressure for sustainability, environmental stewardship, and nutritionally dense products, novel approaches such as bacterial modification of plant growth are one such avenue. Taking a page from the well established and agronomically successful incorporation of Rhizobia in soybeans, new species of plant endophytes are being employed outside of their traditional hosts. One potential endophyte, Gluconacetobacter diazotrophicus, has shown promise in recent trials. Not only can this bacterium non-pathogenically infect myriad agriculturally important crops, it has shown the capability to fix atmospheric nitrogen, produce plant growth promoting hormones such as IAA, GA1 and GA3, and increase plant water use efficiency (WUE).

This project aims to explore the inoculation of two agronomically important fruit crops in the Northeastern US, tomatoes and peppers, by the bacterium G. diaz. Tomatoes represent a high value crop that has come under attack in recent years for nutrient dilution at the expense of yield, as well as high exogenous fertilization requirements. Peppers are a very high value crop that are increasingly being sown in controlled environments to ensure year round fresh availability, and as with tomatoes require extensive, unsustainable fertilization regimes. This research hopes to examine meeting the needs of production while limiting the concomitant costs, both financial and environmental, through replacement of exogenous N fertilization viz bacterial inoculation. Simultaneously, we anticipate assessing potential increases of production over standard practices, and any effected secondary metabolite changes in the produced fruits from bacterially sourced plant growth regulating hormones (PGRH). If even one of these three results can be positively achieved, the benefit to crop production for northeast farmers invested in these two crops could be substantial. Equally important, as bacterially sourced nitrogen is ‘loss-proof’ and may decrease exogenous N requirements, the environmental benefits could lead to greater sustainability

Research

Materials and methods:

To assess inoculation effects of G. diazotrophicus on C. annuum, a factorial experiment combining 6 levels of nitrogen fertilization and 2 levels of bacterial presence began on 10/25/2018. All experimental units were self-contained in closed system Deep Water Culture (DWC) hydroponic buckets, purposely designed and built for this experiment. Each bucket was of the standard 5 gallon (~ 18L) size and was opaque to prevent algal growth in the root zone. Externally, each bucket had a clear plastic standpipe for rapid assessment of water levels inside the DWC, as well as to expedite drainage. Each DWC was lidded with an opaque lid, again to prevent light penetration. Each lid had a 3″ hole for the plant growth netcup basket, centrally drilled, and three small holes; two proximal to the standpipe and one distal. The first proximal of these, ~3/4″ in diameter, was used for nutrient delivery and EC/pH measurements, and was stoppered with a #2 rubber stopper. The next proximal hole, ~1/4″ in diameter, was uncovered and served as an air pressure equalization port. The remaining hole was an entry port for a 3/16″ ID, 1/4″ OD air hose used to deliver aeration to the nutrient solution, ensuring root zone oxygenation. The air hose delivered approximately 10 L of ambient air per minute, ensuring oxygen saturation of the solution surrounding the roots.

In order to assess potential exogenous nitrogen replacement potential, five levels of nitrogen varied from a CEAC recommended level of 190 ppm. Deficient levels were 25% of Recommended (%R), ~47.5 ppm N; 43.75 %R, ~83 ppm N; and 62.5 %R, ~119 ppm N.  A suboptimal level of 81.25 %R was included at ~154 ppm N, as well as a supra-optimal level of 118.75 %R, ~226 ppm N. The control was the 100 %R, ~190 ppm N recommendation. Equal gradation will allow for regression if appropriate.

In order to assess inoculation effect, two levels of bacterial presence were incorporated, as ‘Inoculated’ or ‘Virgin’. ‘Inoculated’ seeds were treated with a surface wash and soak in Gluconacetobacter diazotrophicus in an adjuvant solution. ‘Virgin’ seeds were treated with a G. diaz free dH2O wash and soak. 50 randomly assigned seeds were inundated with 200 ul of respective solution in a 60 mm sterile petri dish, and periodically swirled to ensure full exposure. Both treatments lasted for 10 minutes, after which excess liquid was drained and the seeds allowed to air dry at room temperature (~ 21 oC) for 14 hours. (The adjuvant solution is a proprietary mix of stickeners and bacterial support components, whose exact composition is protected under an NDA.)

The bacteria for this experiment was cultured in sterilized liquid ATGUS media [yeast extract (2.7 g/l), glucose (2.7 g/l), mannitol (1.8 g/l), MES buffer (4.4 g/l, K2HPO4 (0.65 g/l), pH 6.5], and incubated for five days at 60 rpm and 30oC. CFU was determined by serial dilutions and plating in petri dishes (ATGUS solid media [agar (0.8% w/v), yeast extract (2.7 g/l), glucose (2.7 g/l), mannitol (1.8 g/l), MES buffer (4.4 g/l, K2HPO4 (0.65 g/l), pH 6.5]) as well as OD600 spectroscopy (0.6). The applied CFU was 2.4e7/ml.

Seeds were sown in tared 1.5″ AO36/40 rockwool cubes (Grodan, Inc.) with 2 seeds placed per pre-drilled hole. A small plug of rockwool was pressed into the hole, over the seed, to ensure contact. Seeds were germinated in a Growth Chamber (Conviron Systems, Inc.), 16h at 25oC, 8h at 22oC, until seedings emerged. Plants were provided only with tap water to maintain moisture of rockwool; no nutrient solution was added. Upon emergence, a 16h ‘day’ (PAR ~220 umol, 25oC) and 8h ‘night’ (darkness, 22oC) was maintained until roots began to penetrate the rockwool base. Again, only N-free tap water was provided at this time. Upon root emergence, the plants were randomly assigned to individual DWC buckets according to inoculation status, were seated in 3″ rockwool blocks seated in 3″ round plastic netcups, and placed into the predrilled bucket lid holes. After 24 hours in situ, plants were thinned to a single plant per DWC.

Greenhouse conditions were maintained to within acceptable growing parameters, PAR ~220-236 @ 16h day, 8h night; Temperature 62oC-74oC; humidity ambient. Nutrient solution changes will occur and harvesting will occur as necessary within the normal timeframes.

Trial List:

Pepper, sweet (Capsicum annuum), Two (2) trials

Pepper, piquant (Capsicum annuum var. Jalapeno Early), Two (2) trials

Tomato, determinate (Solanum lycopersicum var. BHN 589), Two (2) trials

Tomato, indeterminate (Solanum lycopersicum var. Rebelski), Two (2) trials*

*Note: Indeterminate tomatoes were abandoned in favor of a quicker turnaround piquant pepper due to delays in funding

Files:

Tomato-Layout  Greenhouse Placard for Tomato Round #1, #2

SebringSeminar2019 Presentation of Preliminary results as part of Penn State Plant Science Spring Seminar Series 

Current project status, January 2020. 

Round #1 of Pepper(sweet) trial 100% complete, agronomic. 45% complete, analytical. Plant sown 10/26/2018, fruit harvested (by batch) 2/26/2019-3/5/2019. Whole plant (Veg APP/BPP/TPP) harvested 3/10/2019-3/15/2019. 100% of vegetative, 95% of fruit dried and/or freeze dried for TKN and C/N combustion Nitrogen analysis. Fruit remainder 5%, representative sample 3x ~ 50g per plant, frozen awaiting HPLC nutritive analysis.  Initial results show increased total plant biomass with inoculation (Inoc) (p<0.01), increased Aerial Plant Production (APP) with Inoc (p<0.01), increased fruit number per plant w/ Inoc (p<0.05), suggestive increased fruit production with Inoc (p<0.1) Preliminary conclusion is that Inoculation effects less fruit abortion/more fruiting body growth/retention; Increased plant biomass overall; Increased APP. Residual effluents assessed for residual N; to be combined with plant and fruit Total N for mass balancing once HPLC samples run and returned.

Metrics Measured, per plant:

# Peppers, # Grade A (normal) Peppers, # Off-grade (abnormal) Peppers, Weight per pepper (g), Total fruit weight (g), Dried fruit weight (g), Total Aboveground Plant Production, vegetative (APP) (g), Total Belowground Plant Production, vegetative (BPP) (g), Dried Matter APP (g), Dried Matter BPP (g)

To be measured: N mass balance. Phenolics & secondary metabolites of fruit (vitamins, minerals, etc…) Market value of produced fruit.

Round #1 of Pepper(piquant) trial 100% complete, agronomic. 40% complete, analytical. Plant sown 4/13/2019, fruit harvested (by batch) 8/4/2019, 8/20/2019. Whole plant (Veg APP/BPP/TPP) harvested 8/23/2019. 100% of vegetative, 90% of fruit dried and/or freeze dried for TKN and C/N combustion Nitrogen analysis. Fruit remainder 10%, representative sample 2x ~ 25-40g per plant, frozen awaiting HPLC nutritive analysis.  Initial results show increased Aerial Plant Production (APP) with Inoc (p<0.1), increased fruit number per plant w/ Inoc (p<0.05), decreased fruit size with Inoc (p<0.1) Preliminary conclusion is that Inoculation effects less fruit abortion/more fruiting body growth/retention; Increased APP; no effect on BPP. Residual effluents assessed for residual N; algal contamination from delay between harvest and assessment due to equipment backlog may impair N mass balancing efforts.

Metrics Measured, per plant:

# Peppers, # Grade A (normal) Peppers, # Canning/Corked (abnormal) Peppers, Corked/Grade A ratio, Weight per pepper, indiv. (g), Total combined fruit weight (g), Dried fruit weight, indiv. and combined (g), Total Above-ground Plant Production, vegetative (APP) (g), Total Below-ground Plant Production, vegetative (BPP) (g), Dried Matter APP (g), Dried Matter BPP (g)

To be measured: N mass balance (possibly). Secondary metabolites, vitamins, minerals, capsaicinoids and dihydrocapsaicinoids. Market value of produced fruit.

Round #1 of Tomato trial 100% complete, agronomic. 40% complete, analytical. Plant sown 4/13/2019, fruit harvested (by batch) 7/16/2019, 7/24/2019, 8/2/2019, and 8/11/2019. Whole plant (Veg APP/BPP/TPP) harvested concurrently with last fruit harvest 8/11/2019. 100% of vegetative, 90% of fruit dried and/or freeze dried for TKN and C/N combustion Nitrogen analysis. Fruit remainder 10%, as available per plant when harvest weight of individual fruit > 150g, frozen awaiting HPLC nutritive analysis.  Total samples collected for HPLC ~360 fruit. Inoculation status from frozen fruit not yet completed, awaiting HPLC instrumentation availability before thawing. Initial results show increased total plant biomass with inoculation (Inoc) (p<0.05), increased Aerial Plant Production (APP) with Inoc (p<0.05), increased percentage of fruit showing aberration w/ Inoc (p<0.1), increased early fruit maturation without Inoc (p<0.1), increased later fruit production with Inoc (p<0.1). Preliminary conclusion is that Inoculation effects delayed fruit maturation and exacerbates known fruit problems, such as blossom end rot; Increased plant biomass overall; Increased APP. Residual effluents assessed for residual N; to be combined with plant and fruit Total N for mass balancing once HPLC samples run and returned.

Metrics Measured, per plant:

# Ripe Tomatoes (all harvests), # Unripe Tomatoes (final harvest), # Grade A (normal) Fruit, # Off-grade (abnormal) Fruit, # Diseased fruit, Weight per fruit, indiv., fresh (g), Total combined fresh fruit weight (g), Dried fruit weight, indiv. and total (g), Dried Matter APP (g), Dried Matter BPP (g)

To be measured: N mass balance. Flavenoids, vitamins, minerals, nutritive components, lutein and other tomato specific secondary metabolites. Market value of produced fruit.

Round #2 of Tomato trial. Delayed. Trial started 10/13/2019 as only a single set of growth materials (buckets, pumps, etc) and greenhouse space was available for the project (PSU ASI GH112). Currently plants are ~ 18″ tall, anticipatory first harvest early Spring 2020.

Round #2 of Pepper(piquant) trial. Delayed.  Trial started 10/18/2019 as only a single set of growth materials (buckets, pumps, etc) and greenhouse space was available for the project (USDA GH 1B, 1C). Currently plants are ~ 9″ tall, anticipatory first harvest early/mid Spring 2020.

Round #2 of Pepper(sweet) trial. Delayed. Potentially not able to be completed with current time-frame. Not necessary for original scope of project.

Research results and discussion:

TBC

Research conclusions:

TBC

Participation Summary
1 Farmer participating in research

Education & Outreach Activities and Participation Summary

1 Webinars / talks / presentations

Participation Summary

10 Farmers
25 Number of agricultural educator or service providers reached through education and outreach activities
Education/outreach description:
Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.