DOI: 10.5061/dryad.ttdz08m85
title: Dataset: Method and timing of rhizobacteria inoculation to plant roots alters success and strength of aphid suppression

description: Insect pests cause substantial yield losses globally, necessitating novel
 pest control approaches beyond chemical pesticides. Rhizobacteria,
 beneficial root-associated bacteria, present a promising alternative by
 enhancing plant growth and defences against pests. This study explored the
 use of sodium alginate encapsulation for precise inoculation of two
 rhizobacteria, Acidovorax radicis and Bacillus subtilis, to
 suppress aphid (Sitobion avenae) populations on barley (Hordeum vulgare).
 We optimized a method using 4mm diameter wet-matrix alginate beads for
 controlled bacterial delivery directly to plant roots. Rhizobacteria
 inoculation via alginate beads at root emergence significantly reduced
 aphid reproductive output, outperforming seed soaking methods, with
 increased effects when plants were infested with a high initial density of
 aphids. This suggests the prolonged release from alginate beads led to
 more consistent bacterial establishment to enhance plant defence responses
 triggered by aphid feeding. Alginate bead inoculation demonstrated
 effective long-term bacterial viability following storage at 4° C for
 eight months. Our findings highlight the potential of alginate
 bead-encapsulated rhizobacteria for reducing aphid populations on barley
 but emphasizing the need for precise inoculation timing and placement.
 This approach offers a robust method for empirical research and practical
 agricultural application, paving the way for sustainable pest management
 strategies. Future work should focus on optimizing formulations and
 understanding plant-microbe interactions to enhance field efficacy.
description: This dataset includes results from experiments investigating the
 effects of root-inoculated bacteria on barley plants (<em>Hordeum
 vulgare</em>) and their interactions with aboveground aphid
 herbivores (<em>Sitobion avenae</em>). Two bacterial species,
 <em>Acidovorax radicis</em> N35 and <em>Bacillus
 subtilis </em>B171, were used, with a control treatment of 10mM
 MgCl2. Plants of two barley varieties (Barbarella and Firefoxx) were grown
 in Levington’s Advance F1 low nutrient compost. Bacterial cultures were
 prepared by growing on nutrient agar or broth, harvested, and resuspended
 at OD600=2.0. Barley seeds were surface-sterilized and inoculated either
 by soaking in bacterial solutions or embedding bacteria in alginate beads
 formed by dropping bacterial-alginate mixtures into CaCl2. 
 Three fully-factorial experiments were conducted. The first
 compared seed soaking and bead inoculation methods on aphid numbers,
 measuring plant shoot and root length and aphid reproduction after two
 weeks. The second tested timing of bead inoculation relative to seed
 germination and aphid infestation, tracking aphid fecundity and adult
 survival over 30 days. The third experiment examined the effect of
 different initial aphid densities on plant growth and aphid population
 dynamics. All experiments were arranged in randomized complete block
 designs and conducted in glasshouse or controlled growth cabinet
 conditions. This dataset provides detailed measurements
 of plant growth (shoot and root length), aphid reproduction and survival,
 offering insights into plant-microbe-insect interactions.
description: # Dataset: Method and timing of rhizobacteria inoculation to plant roots
 alters success and strength of aphid suppression
 [https://doi.org/10.5061/dryad.ttdz08m85](https://doi.org/10.5061/dryad.ttdz08m85) ## *Principle Investigator Contact Information* ``` Name: Dr Sharon Zytynska Institution: University of Liverpool, UK Email: sharon.zytynska@liverpool.ac.uk ``` ## Description of the data and file structure Data from three experiments is included in this submission, across five .csv files. Each experiment is fully factorial, with columns detailing the treatments used. **Experiment 1:**Blenkinsopp_Expt_1_data.csv [contains all data needed to run associated R code] **Experiment 2:**  Blenkinsopp_Expt_2_data_totals.csv [data in wide-format, for analysis of totals] Blenkinsopp_Expt_2_data_extended.csv [data in long-format, for analysis over time] Blenkinsopp_Expt_2_data_survival.csv [data on survival for survival analyses] **Experiment 3:** Blenkinsopp_Expt_3_data.csv [contains all data needed to run associated R code] ## Files and variables The tables below give explanation of the column headings and units for each dataset. **Experiment 1:**  Blenkinsopp_Expt_1_data.csv | Column | Entry | unit | Explanation | | :----- | :--------------- | :---------- | :--------------------------------------------------------------------- | | A | Barley | name | Barley variety used | | B | number.inoc | number | Single or double inoculation | | C | type.inoc | name | Seed, bead or double (seed and bead) inoculation | | D | Bacteria | name | Bacterial treatment, A. radicis, B. subtilis or control | | E | Bacteria.seed | name | Seed treatment | | F | Bacteria.bead | name | Bead treatment | | G | rep | number | Replicate | | H | Tray | number | Tray (nested by bacteria treatment) | | I | Pot | number | Pot number | | J | root.d6 | number (cm) | Root length at transplantation | | K | shoot.d6 | number (cm) | Shoot length at transplantation | | L | aphids.on.d9 | number | Number of aphids added on day 9 | | M | adult.aphids.d23 | number | Number of adult aphids counted on day 23 (14 days of aphid feeding) | | N | offspring.d23 | number | Number of juvenile aphids counted on day 23 (14 days of aphid feeding) | | O | shoot.d23 | number (cm) | Root length on day 23 | | P | root.d23 | number (cm) | Shoot length on day 23 | | Q | aphid.number | number | Total number of aphids | | R | control.ave | number | Average number of aphids for control pots | | S | relative | number | Relative number of aphids on inoculated vs control | **Experiment 2:**  Blenkinsopp_Expt_2_data_totals.csv | Column | Entry | unit | Explanation | | :----- | :---------------------------- | :--------------- | :------------------------------------------------------------------------------------------ | | A | Bacteria | name | Bacteria treatment | | B | Bacteria.PA | presence/absence | presence/absence of bacteria | | C | Timing | name | Timing treatment | | D | Inoc\_timing | name | Combined factor of control and inoculation by time | | E | rep | number | replicate | | F | Tray | number | Tray number | | G | Pot | number | Pot number | | H | total.offspring | number | Total number of offspring | | I | repro.days | number | Number of aphid reproduction days | | J | Rel\_ave\_total.off | number | Relative number of aphids on inoculated vs control | | K | daily.offspring.by.surv.adult | number | Number of offspring produced daily per suriving adult | | L | Shoot.d0 | number | shoot height, day 0 | | M | Shoot.d1 | number | shoot height, day 1 | | N | Shoot.d4 | number | shoot height, day 4 | | O | Shoot.d8 | number | shoot height, day 8 | | P | Shoot.d11 | number | shoot height, day 11 | | Q | Shoot.d15 | number | shoot height, day 15 | | R | Shoot.d18 | number | shoot height, day 18 | | S | Shoot.d22 | number | shoot height, day 22 | | T | Shoot.d25 | number | shoot height, day 25 | | U | Shoot.d29 | number | shoot height, day 29 | | V | O.aphid.d1 | number | number of offspring, day 1 | | W | O.aphid.d4 | number | number of offspring, day 4 | | X | O.aphid.d8 | number | number of offspring, day 8 | | Y | O.aphid.d11 | number | number of offspring, day 11 | | Z | O.aphid.d15 | number | number of offspring, day 15 | | AA | O.aphid.d18 | number | number of offspring, day 18 | | AB | O.aphid.d22 | number | number of offspring, day 22 | | AC | O.aphid.d25 | number | number of offspring, day 25 | | AD | O.aphid.d.29 | number | number of offspring, day 29 | | AE | max.offspring | number | Maximum offspring counted in one measurement day | | AF | day.of .max | number | Day of maximum reproduction | | AG | A.aphid.d1 | number | number of adult aphids, day 1 | | AH | A.aphid.d4 | number | number of adult aphids, day 4 | | AI | A.aphid.d8 | number | number of adult aphids, day 8 | | AJ | A.aphid.d11 | number | number of adult aphids, day 11 | | AK | A.aphid.d15 | number | number of adult aphids, day 15 | | AL | A.aphid.d18 | number | number of adult aphids, day 18 | | AM | A.aphid.d22 | number | number of adult aphids, day 22 | | AN | A.aphid.d25 | number | number of adult aphids, day 25 | | AO | A.aphid.d29 | number | number of adult aphids, day 29 | | AP | adult.days | number | Number of days with surviving adults | | AQ | longevity | number | avergae longevity of the two adult aphids | | AR | offspring.by.surv.adult | number | Number of offspring per surviving adult | | AS | O.surv.Ad.d1 | number | number of offspring divided by adult aphids, day 1 | | AT | O.surv.Ad.d4 | number | number of offspring divided by adult aphids, day 4 | | AU | O.surv.Ad.d8 | number | number of offspring divided by adult aphids, day 8 | | AV | O.surv.Ad.d11 | number | number of offspring divided by adult aphids, day 11 | | AW | O.surv.Ad.d15 | number | number of offspring divided by adult aphids, day 15 | | AX | O.surv.Ad.d18 | number | number of offspring divided by adult aphids, day 18 | | AY | O.surv.Ad.d22 | number | number of offspring divided by adult aphids, day 22 | | AZ | O.surv.Ad.d25 | number | number of offspring divided by adult aphids, day 25 | | BA | O.surv.Ad.d29 | number | number of offspring divided by adult aphids, day 29 | | BB | Daily.offspring.per.adult.d1 | number | number of offspring divided by adult aphids and number of days between observations, day 1 | | BC | Daily.offspring.per.adult.d4 | number | number of offspring divided by adult aphids and number of days between observations, day 4 | | BD | Daily.offspring.per.adult.d8 | number | number of offspring divided by adult aphids and number of days between observations, day 8 | | BE | Daily.offspring.per.adult.d11 | number | number of offspring divided by adult aphids and number of days between observations, day 11 | | BF | Daily.offspring.per.adult.d15 | number | number of offspring divided by adult aphids and number of days between observations, day 15 | | BG | Daily.offspring.per.adult.d18 | number | number of offspring divided by adult aphids and number of days between observations, day 18 | | BH | Daily.offspring.per.adult.d22 | number | number of offspring divided by adult aphids and number of days between observations, day 22 | | BI | Daily.offspring.per.adult.d25 | number | number of offspring divided by adult aphids and number of days between observations, day 25 | | BJ | Daily.offspring.per.adult.d29 | number | number of offspring divided by adult aphids and number of days between observations, day 29 | **Experiment 2:**  Blenkinsopp_Expt_2_data_extended.csv | Column | Entry | unit | Explanation | | :----- | :---------------------- | :--------------- | :------------------------------------------------- | | A | Bacteria | name | Bacteria treatment | | B | Bacteria.PA | presence/absence | presence/absence of bacteria | | C | Inoculation | name | Timing treatment | | D | Inoc\_comb\_control | name | Combined factor of control and inoculation by time | | E | rep | number | replicate | | F | Tray | number | Tray number | | G | Pot | number | Pot number | | H | Day | number | Total number of offspring | | I | Shoot | number (cm) | shoot length | | J | Offspring | number | Number of offspring | | K | Adults | number | Number of Adults | | L | cumulative.Off | number | Cumulative number of offspring | | M | Daily.offspring | number | Daily offspring | | N | offspring.by.surv.adult | number | Daily offspring per surviving adult | **Experiment 2:**  Blenkinsopp_Expt_2_data_survival.csv | Column | Entry | unit | Explanation | | :----- | :------------------ | :--------------- | :------------------------------------------------- | | A | Bacteria | name | Bacteria treatment | | B | Bacteria.PA | presence/absence | presence/absence of bacteria | | C | Inoculation | name | Timing treatment | | D | Inoc\_comb\_control | name | Combined factor of control and inoculation by time | | E | rep | number | replicate | | F | Tray | number | Tray number | | G | Pot | number | Pot number | | H | Aphid\_num | number | Adult 1 or 2 | | I | Fecundity | number | Number of offspring produced | | J | Fecund\_real | number | Number of offspring produced, per adult | | K | Death\_day | number | Day of death of aphid | **Experiment 3:**  Blenkinsopp_Expt_3_data.csv | Column | Entry | unit | Explanation | | :----- | :-------------- | :--------------- | :----------------------------------------------------------------- | | A | Barley | name | Barley variety used | | B | Bacteria | name | Bacteria treatment | | C | Bacteria.PA | presence/absence | Bacteria presence/absence | | D | Aphid | name | Aphid initial density, low (1 adult) or high ( 3 adults, 3 nymphs) | | E | rep | number | replicate | | F | Tray | number | Tray number | | G | Pot | number | Pot number | | H | shoot.d0 | number (cm) | shoot height day 0 | | I | shoot.d2 | number (cm) | shoot height day 2 | | J | aphids.on.d2 | number | Number of adults added on day 2 | | K | adult.d4 | number | Number of adults on day 4 | | L | wing.d4 | number | Number of winged aphids on day 4 | | M | offspring.d4 | number | Number of juvenile aphids on day 4 | | N | total.d4 | number | Total number of aphids on day 4 | | O | d4.cont | number | Average number of aphids for control pots | | P | d4.rel | number | Relative number of aphids on inoculated vs control | | Q | SD.C.d4 | number | Standard deviation of control pots | | R | effect.size.d4 | number | Effect size estimation | | S | shoot.d7 | number (cm) | shoot height day 7 | | T | shoot.d10 | number (cm) | shoot height day 10 | | U | adult.d10 | number | Number of adults on day 10 | | V | offspring.d10 | number | Number of juvenile aphids on day 10 | | W | total.d10 | number | Total number of aphids on day10 | | X | d10.cont | number | Average number of aphids for control pots | | Y | d10.rel | number | Relative number of aphids on inoculated vs control | | Z | SD.C.d10 | number | Standard deviation of control pots | | AA | effect.size.d10 | number | Effect size estimation | ## Code/software R code included in submission, hosted by Zenodo Bead_inoculation_paper_R_code_June2025.R