Effects of crop diversification and mulching on pest abundance in Brassica oleracea L. var. Italica.Connor Sanit, Terra Rose, Emily Shiue, Sean Go, and Melissa Makous

IntroductionThe current dominant agricultural regime is characterized by large-scale monocultures that use excessive external inputs and other industrial-style practices driven by capitalist motives. Large transnational corporations promote conventional agriculture practices that unfortunately create vast environmental degradation through soil depletion, biodiversity loss, and pollution of natural resources (Altieri, 2007). Conventional growers and the corporations running these operations consider these consequences as externalities imposed on the greater population and threatening environmental and public health. External inputs (synthetic fertilizers, chemical pesticides, etc.) used in production pollute our watershed and emit greenhouse gases indiscriminately. This is simply not a sustainable path for agriculture production. Our research seeks to promote an alternative to industrial agriculture that considers biodiversity and soil fertility effects on pest management and yield. We planted Brassica oleracea L. var. italica (broccoli) in four groups, each with a different intercrop including one monoculture control group. The three intercrops were vicia faba (fava bean), fagopyrum esculentum (buckwheat), and brassica (mustard). Additionally, half of each group was covered in straw mulch to study how mulch usage affects results as well. Our intent was to show how each factor affected pest populations, natural enemy abundance, and plant development. Using intercrops and mulch for pest control is an alternative to harmful inputs used on a wide scale today. These techniques increase in-situ biodiversity and are thus more resistant to pest and disease outbreaks.

Previous StudiesThe idea that intercropping can be used as a biological approach to pest management has been studied since Pimentel (1961) scientifically proved that crop diversification can lead to lower pest incidence. Many brassica plants have been used as a basis for these studies including collards (Root 1973) and brussel sprouts (Tukahirwa & Coaker 1982). Broccoli was used by Ponti et al. (2007) to test this idea using buckwheat and mustard as companion plants. They additionally tested the broccoli in both competitive and noncompetitive cropping systems and with chemical and organic fertilizers to show differences in resulting biomass. In this study, the only factors we consider to affect yield are intercropping and mulch. We also decided to include a leguminous companion plant, vicia faba (fava bean) in this experiment, which can increase soil fertility, a method used in lieu of application of any soil enriching fertilizers. Previous studies have shown how living leguminous mulches have beneficial effects on pest populations in brassicas. (Andow et al. 1986, Costello 1993). By using a leguminous intercrop (in addition to buckwheat and mustard) in conjunction with a non-living mulch we have taken different components of previous studies and combined them in order to adapt this experiment to specific

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environmental conditions in California.

BroccoliThe state of California produces about 90% of the entire United States broccoli using the conventional monoculture, high input industrial model (Sances & Ryn 1999). Identifying intercrop species that reduce harmful pests and increase beneficial predators could decrease pesticide application and inform farmers of land management practices that are more ecologically responsible. This research goes beyond the scope of biodiversity to try to understand functional biodiversity, and how specific intercrop treatments or mulch can add to the functionality of the agroecosystem. We can then identify which theory best explains the results: natural enemies hypothesis, resource concentration hypothesis, and/or plant vigor hypothesis. The “natural enemies” hypothesis predicts that increasing plant biodiversity will provide more habitat for beneficial predators that will attack and kill more pests. The “resource concentration hypothesis” predicts that having more crop diversity will inhibit pest population growth as it is more difficult to find their preferred crop. Lastly, the “plant vigor hypothesis” predicts that nutritious high vigor plants will attract more pests while low vigor plants, which may not produce naturally occurring defense compounds, will also attract more pests. Each cropping system impacts pest control in different ways: buckwheat produces nectar and pollen which attracts beneficial insects, fava bean increases soil fertility by fixing nitrogen in the atmosphere, mustard can act as a trap crop for pests, and mulch provides habitat/shelter for key predators (Altieri and Wilson, 2014). The experiment will test which treatment can best provide biological control of known broccoli pests, as well as produce healthier soil for better plant growth.

MulchThe decision to include straw mulch in this study was affected by current climatic conditions in California. With the severe drought in place, mulch plays a key role in retaining soil moisture (Thompson and Hoffmann, 2007). Traditional monocultures are designed for plots with idealized conditions. The ongoing drought challenges these conditions, making it is necessary for us to research and pose alternative land practices in order to deal with environmental stress. Further, mulch has the ability to reduce weed and pest population growth, protect from wind and water erosion, enhance soil fertility and structure, and reduce soil compaction (Costello & Altieri 1995). Thompson and Hoffmann (2007) used straw mulch in vineyards to study its potential as habitat for beneficial predators and as a water conservation method. They found that pest abundance did not increase while natural enemies such as spiders, beetles, and predatory bugs, did increase. Our research hopes to find quantitative proof supporting the benefits of using a mulch for both biological control and water conservation.

PestsIn our study we focused on two main pests: Brevicoryne brassicae (cabbage aphids) and Phyllotreta cruciferae (crucifer flea beetles). Cabbage aphids are common pests that attack the

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mustard family by feeding on the phloem in plant tissue using their stylets (Altieri et al. 2007). This can greatly reduce yields. We identified aphids at three different stages in the growth cycle: apterous (non-winged), alate (winged), and parasitized. The crucifer flea beetle is an invasive insect that emerges once a year. After the adult flea beetle lay their eggs in the spring, the larvae reside in the soil feeding on roots until they mature into adults around August. This new generation of adults then feed on the leaves leaving small holes.

MethodsThis field research took place at the Oxford Tract, an open field planting space for research at the University of California, Berkeley. We evaluated the performance of broccoli polyculture relative to a broccoli monoculture.The broccoli polyculture used three intercrop treatments. The experimental area was divided into twelve plots, with eight cropping systems, each replicated three times: broccoli monoculture, with or without mulch; broccoli intercropped with buckwheat, with or without mulch; broccoli intercropped with fava, with or without mulch; and broccoli intercropped with mustard, with or without mulch.Pictured below is how the different treatments were subdivided and replicated on the plot.

Figure 1. Map of the experiment plots.Plot ManagementOn July 21, 2014 the broccoli seeds were sowed in a greenhouse. Then, on August 23, the seedlings were moved to the plots and planted at a density of 1 plant every 50 cm within each row. When planted, each seedling received ~200 cm3 of compost from the City of Berkeley

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green waste recycling program. Each intercrop treatment was then sown in its appropriate plot. On September 3rd, the plots were weeded and mulch was added. Drip irrigation was used every 2-3 days for 3 hours to maintain the plant growth. Every week, the plots were weeded and where the intercrops were outgrowing the broccoli, they were trimmed. This continued until the broccoli became fully grown after which point they were harvested.

Data CollectionData on plant development and insect populations was collected weekly in each of the twelve plots for six weeks. A computer-program generated a random sample of ten plants--five treated with mulch, five untreated—each week. In order to ensure there are no “spillover” effects from neighboring plots using different treatments, all plants located in the outside rows (Rows 1 and 5), as well as the first and last two plants in any given row (plants 1-2 and 11-12) were omitted. Figure 2 provides an illustration of plants eligible to be sampled. Additionally, plants that visually appeared abnormal (e.g., shorter than neighboring plants, visibly dead, etc.) were omitted and an adjacent plant in the same row was measured instead to control for unknown factors affecting growth.

Figure 2. Map of Typical Experimental Plot. Data was not collected for plants in the grey-shaded area. All experimental data used for this experiment comes from plants located in the white-shaded area.

Plant development was measured by: (1) plant height, as measured in centimeters from the base of the plant to the top of the broccoli crown; and (2) number of fully expanded and mature leaves, ignoring any secondary leaves. The pest insect population present on each sample plant was captured by counting and recording on three of five randomly chosen fully mature leaves, the (1) number of apterous-stage aphids (Brevicoryne brassicae); (2) number of alate-stage aphids; and (3) number of parasitized aphids, also known as “mummy” aphids. For each of the plants in the sample, the parasitism rate was calculated by dividing the total number of parasitized aphids by the total number of all aphid types. Other pests and beneficial insects were measured weekly using yellow sticky traps in both the mulch and no mulch areas of the plots. These traps captured the (1) number of flea beetles (pest); (2) number of ladybird beetles (beneficial); and (3) number of syrphid flies (beneficial). Finally, yield biomass was measured during the last couple weeks of data collection by (1) root weight, measured in the last week; (2) plant weight, for the last week; and (3) crown weight, for

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the last 2-3 weeks. AnalysisThe data points above were used in two different comparisons. One comparison measured the influence of intercrops, using the data points collected for each of the three intercropped groups to compare against the control (broccoli monoculture) and each other. The other comparison measured the influence of mulch, using the data points for the treatments of mulch versus no mulch in all of the plots. These data points were analyzed using a statistical analysis site StatWing.com that calculated analysis of variance (ANOVA), returning the p-values for each comparison, an indicator for statistical significance in data. The mean and standard error of mean were also calculated for each data set and were compiled into the results, represented as graphs, below.

ResultsThis section contains graphs only for which a statistically significant relationship was found. For data visualizations of all other data points, please refer to Appendix A.

For this experiment, a 95% confidence level was selected and calculated p-values were compared to the significance level of 0.05. Where p > 0.05, then we failed to reject the null hypothesis H0, meaning there is no significant difference between the treatment groups tested. Where p < 0.05, we reject the null hypothesis H0, meaning there is a significant difference between the treatment groups tested.

Pest Response

There was a statistically significant difference between the average flea beetle presence in the buckwheat (B) and mustard (A) treatment groups, and between fava (B) and mustard (A) treatment groups at the 95% significance level. [Buckwheat-Mustard p = 0.0101; Fava-Mustard p = 0.0136]. (Fig. 3)

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Figure 3. Average flea beetle presence as influenced by intercrop treatment.

There was a statistically significant difference in the average number of parasitized aphids between the buckwheat (C) and fava (B), between buckwheat (C) and monoculture (A), between fava (B) and monoculture (A) at the 95% significance level. [Buckwheat-Fava p = 0.00156; Buckwheat-Monoculture p = 0.0363; Fava-Monoculture p < 0.001]. (Fig. 4)

Figure 4. Average number of parasitized Aphids as influenced by intercrop treatment.

Additionally, we found that there is a statistically significant difference between the average parasitism rate between the buckwheat (A) and fava (B), and between monoculture (A) and fava (B) at the 95% significance level. [Buckwheat-Fava p = 0.00277; Fava-Monoculture p = 0.0128]. (Fig. 5)

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Figure 5. Parasitism rate as influenced by intercrop treatment.

We also found a statistically significant difference between the average presence of ladybirds between buckwheat (A) and mustard (B). [Buckwheat-Mustard p = 0.0131]. There was no statistically significant difference among the average presence of syrphid flies. The graphs were combined for comparison (Fig. 6)

Figure 6. Presence of ladybirds and syrphid flies as influenced by intercrop.

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Figure 7. Broccoli yield biomass measured by fresh head weight and as influenced by intercrop treatment.

Above shows that there is a significant difference in average plant crown weight between buckwheat and monoculture, fava and mustard, and monoculture and mustard.[Buckwheat-Monoculture p = 0.00434; Fava-Mustard p = 0.0420; Monoculture-Mustard p <0.001]

Discussion

Pest ResponseWith an overall p-value of 0.00716 (figure 3), the only case of statistical significance (a p-value of <.05) was found in the intercrop treatments when looking at the presence of flea beetles. The pair groupings of buckwheat and mustard, and fava and mustard, each had a p-value of 0.0101 and 0.0136 respectively. As illustrated with the Tukey labeling system, none of the other pair groupings had any statistical significance (Figure 3). Since flea beetles prefer mustard family host plants (Altieri and Wilson, 2014), it is no surprise the highest average presence of flea beetles were found in the mustard plots compared to the monoculture, fava, and buckwheat plots. Since broccoli is also a member of the mustard family, intercropping broccoli with mustard doubles the amount of preferred host plants and therefore increases the desirability to the flea beetles compared to monoculture alone. This runs counter to the resource concentration hypothesis because instead of diversity breaking up the concentration of host crops in an area, the concentration is actually increased when intercropping mustard and broccoli because they are both part of the mustard family.

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The statistical significance between the fava and mustard can be explained by the resource concentration hypothesis and the plant vigor hypothesis. As stated above, while the intercropped mustard actually increases the concentration of preferred crops, the intercropping of fava dilutes the concentration of crops that the flea beetles prefer. The fava can be thought of as a camouflage mechanism for the broccoli to distract the flea beetles. Fava is also a leguminous crop that fixes nitrogen in the soil during growth and deposits it in the rhizosphere, the area of soil that is directly affected by plant roots (Hauggaard-Nielsen and Jensen, 2003).

The significance between the buckwheat and mustard in flea beetle presence can be explained with the natural enemies hypothesis. Since buckwheat is known to produce nectar and pollen which attracts beneficial insects, we can expect to see lower levels of flea beetles in the buckwheat plots when compared to the mustard and monoculture plots. More beneficial insects means more predation on the flea beetles and decreases in their overall numbers. This is supported in our results (Figure 3 above).

When it came to apterous aphids, alates, and total aphids, there were no statistically significant relationships in comparing the intercropped treatments to each other, and also in comparing the mulch versus non-mulch treatments. In the mulch versus non-mulch comparison, there was no significant relationship for the presence of flea beetles. Those relationships can be found in Figure 14 of the appendix for the intercropped treatments, and Figures 1, 4, 8, and 10 of the appendix for the mulch versus non-mulch treatments.

Natural Enemy AbundanceWhen looking at the results relating to natural enemy abundance, we found a statistically significant difference in the parasitism rate overall as well as the average presences in syrphid flies and ladybird beetles between pairs of intercropping methods. The parasitism rate is calculated by dividing the number of parasitized aphids (“mummies”) by the total number of aphids on the plant, where each factor can influence the rate in different ways. A high number of parasitized aphids indicates the presence of natural enemies of aphids. The parasitism rate has an overall p-value of 0.00107, showing a highly significant difference among the treatments. In particular, there was a significant difference between buckwheat and fava (p = 0.00277) as well as fava and monoculture (p = 0.0128), but none between the rest of the pairs as seen in Figure 5. Fava was used as an intercrop because as a legume, it enhances soil fertility and thus, it was not intended to attract beneficial insects. This can be seen in the fact that fava had a significantly lower rate of parasitism than buckwheat, which has a large amount of pollen and nectar that attracts beneficial insects. The parasitism rate of fava was also lower in comparison to the monoculture group. Additionally, a further reason for the low parasitism rate in fava could be the fact that there were a significantly larger amount of aphids than parasitized aphids, skewing the rate. This can be explained by the plant vigor hypothesis, in which the fava intercropped plants were overly vigorous and creating a central place for pests to populate. The increased fertility of

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the soil by fixing atmospheric nitrogen by the legume may also have caused the broccoli plants to become too vigorous, making it attractive to pests and increasing the total amount of pests on the plants.

The presence of the beneficial insects, the ladybird beetles and syrphid flies, near the plants indicate that there are more resources around to support beneficial insects. The overall average number of ladybird beetles among the different treatments were statistically significant with a p-value of 0.0233, as seen in Figure 6. Mustard produces many flowers that provide good resources for beneficial insects and this is reflected in our data which shows the mustard intercrop with the highest number of ladybirds. Buckwheat was expected to attract many beneficial insects as well, since it has a large amount of nectar and pollen for insects, but it resulted in the lowest amount of ladybirds, so our natural enemies hypothesis may not be correct in regards to buckwheat as an intercrop. The presence of syrphid flies yielded no significant difference in the results and so we were not able to validate any of the three hypotheses.

Plant Development With regard to the plant development data points we collected (plant height, number of leaves, root weight, plant weight, yield/crown weight), the statistically significant findings were limited. Only yield, measured by crown weight, amongst the intercrop treatment groups was found to be significant. The average yield for the monoculture group was higher than the average yield for the mustard and buckwheat treatment groups, but statistically similar to that of the fava treatment group. This suggests that fava could be an appropriate intercrop for commercial broccoli farmers that does not negatively impact yield and that also has benefits in terms of a reduced flea beetle presence. The N-fixation that occurs on the root nodules in the fava may also facilitate plant growth by increasing the amount of bioavailable nitrogen as the roots of the leguminous plant decompose. Hauggaard-Nielsen and Jensen (2003) studied facilitative root interactions between leguminous and non-leguminous intercrops. Though various laboratory studies have shown a significant nitrogen transfer between non-leguminous and leguminous intercrops, they explain that there is a lack of evidence of this from field studies, possibly due to methodological issues. Regardless, this facilitative root interaction may help explain why fava had higher yields than the other intercropped fields. (Fig. 7)

In terms of the other intercrops, our findings suggest that both mustard and buckwheat occupy a similar ecological niche as broccoli and compete for similar resources, thus affecting yield. Because mustard and broccoli are both found in the brassica family, it makes sense that they have similar resource needs.

Mulch versus no mulchThere were no significant differences between the broccoli plantings grown with or without mulch in any of our data.

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Limitations and ImprovementsThere are several potential limitations to this experiment. First, it is possible that the buffers between treatment groups were insufficient and that the results were affected by “spillover” effects from neighboring plots using different treatments. In other words, if the population of natural enemies increased in the buckwheat intercrop plot, some of these predators may also inhabit the monoculture plot. This would skew the results and suggest a higher number of natural enemies may be present in a monoculture than would be under true monoculture conditions. Second, because the data was collected by dozens of different students, it’s possible that the collection was inconsistent and non-uniform, which also could skew the results. Third, the nature of the Oxford Tract is such that there are several other research plots surrounding the experiment plot. It is also possible that these other plantings impacted the presence or absence of pests and natural enemies.

This experiment could be further improved by limiting the number of comparison intercrop groups to be able to focus solely on the effects of each intercrop. Expanding the buffer between treatments to minimize any “spillover” effects would also improvement in results. Furthermore, having fewer people collecting data would ensure greater consistency and accuracy.

Conclusion

Suggestions for future studiesIn order to confirm these results, this experiment could be performed several more times. Our conclusions came from only one data set, and it is important to have temporal diversity and many data sets in order to reduce the variance and unseen variables that may affect a single experiment. Climatic conditions, time of the year, and human error are all factors that can affect any given experiment, and so it should be repeated in order to see the averages and understand any present trend. For examples, a way to test and see if the mustard intercrop did increase the presence of flea beetles compared to the monoculture of broccoli, would be to plant a plot of monoculture broccoli, a monoculture of mustard, and an intercrop of mustard and broccoli. If the intercrop increases the preferred hosts for flea beetles, one could expect to find consistently higher, and statistically significant differences between the intercropped treatments compared with the monocultures. A way to test the natural enemies hypothesis for buckwheat, would be to have a monoculture control group, an intercropped broccoli and buckwheat group, an intercropped broccoli and mustard group, and a polyculture of buckwheat, mustard, and broccoli. If the hypothesis is supported, one should expect to see significantly lower levels of flea beetles in the buckwheat, mustard, and broccoli polyculture when compared to the broccoli and mustard intercrop. This same setup could be used to test the resource concentration hypothesis with fava. Simply replace the buckwheat with fava, and the results would show lower levels of flea beetles in the fava, mustard, broccoli polyculture when compared to just the mustard and broccoli polyculture, if the hypothesis is supported.

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An extended study with plant development would be necessary to confirm resource competition as the explanation for the reduced yield compared to buckwheat. This could be tested in an experiment comparing the yield of buckwheat monocultures with buckwheat intercropped with broccoli.

Because yield is likely to be the greatest concern for commercial broccoli farmers, a logical follow-up experiment would be to replicate the experiment using only monoculture broccoli and broccoli intercropped with fava to see if the yield results hold. Additionally, in the experiment, both mulch and bare plots were weeded by hand which may not be cost-effective in a commercial setting and so it is likely that farmers would turn to herbicide as a solution. Further experimentation around weed growth and control in mulched versus bare plots and the impact on yield would also be worthwhile of future study.

References

Altieri, M.A. (2007). Fatal harvest: Old and new dimensions of the ecological tragedy of modern agriculture. In P. N. Nemetz (Ed), Sustainable resource management, 189-213. Cheltenham, UK: Edward Elgar.

Altieri, M. A., and M. J. Costello (1995). "Living mulches suppress aphids in broccoli." California Agriculture, 48, 24-28.

Altieri, M. and H. Wilson (2014). “Handout for Section.”

Andow, D.A., Nicholson, A.G., Wien, H.C. & Willson, H.R. (1986) Insect population on cabbage

grown with living mulches. Environmental Entomology, 15, 293-299.

Altieri, M. A., Ponti, L. and Guiterrez, A. 2007. “Effects of crop diversification levels and fertilization regimes on abundance of Brevicoryne brassicae (L.) and its parasitization by Diaeretiella rapae (M’Intosh) in broccoli. Agricultural and Forest Entomology, 9, 209-214.

Costello, M.J. (1994) Broccoli growth, yield and level of aphid infestation in leguminous living mulches. Biological Agriculture and Horticulture, 10, 207-222.

Hauggaard-Nielsen, H. and E.S. Jensen. (2003). Facilitative root interaction in intercrops. Plant and Soil, 274, 237-250.

Pimentel, D. (1961) Species diversity and insect population outbreaks. Annals of Entomological

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Society of America, 54, 76-86.

Root, R. B. (1973) Organization of a plant-arthropod association in simple and diverse habitats: the fauna of collards (Brassica oleracea). Ecological Monograph, 43, 95-120.

Sances, F. V. and Van Ryn, M. 1999. “Crop Profile for Broccoli in California”. Rep. San Luis Obispo: Alliance for Alternative Agriculture.

Thompson, L. J. and Hoffmann, A. A. 2007. “Effects of ground cover (straw and compost) on the

abundance of natural enemies and soil macro invertebrates in vineyards.” Agricultural and

Forest Entomology, 9, 173-179.

Tukahirwa, E.M. & Coaker, T.H. (1982) “Effect of mixed cropping on some insect pests of brassicas; reduced Brevicoryne brassicae infestations and influences on epigeal predators and the disturbance of oviposition behaviour in Delia brassicae.” Entomologia Experimentalis et Applicata, 32, 129-140.

Appendix A: Non-statistically significant graphs

Figure 1. Comparison Between Mean Number of Fleabeetles Across Bare and Mulch Treatments

There is no significant difference between the mean number of fleabeetles across bare and mulch treatments

Figure 2. Comparison Between Mean Number of Ladybirds Across Bare and Mulch TreatmentsThere is no significant difference between the mean number of ladybirds across bare and mulch treatments

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Figure 3. Comparison Between Mean Number of Syrphid Flies Across Bare and Mulch Treatments.

There is no significant difference between the mean number of syrphid flies across bare and mulch treatments.

Figure 4. Comparison Between Mean Number of Aphids Across Bare and Mulch Treatments

There is no significant difference between the mean number of aphids across bare and mulch treatments

Figure 5. Comparison Between Mean Parasitism Rate Across Bare and Mulch Treatments

There is no significant difference between the mean parasitism rate across bare and mulch treatments

Figure 6. Comparison Between Mean Number of Leaves Across Bare and Mulch Treatments

There is no significant difference between the mean number of leaves across bare and mulch treatments

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Figure 7. Comparison Between Mean Plant Height Across Bare and Mulch Treatments

There is no significant difference between the mean plant height across bare and mulch treatments

Figure 8. Comparison Between Mean number of Alates Across Bare and Mulch Treatments

There is no significant difference between the mean number of alates across bare and mulch treatments

Figure 9. Comparison Between Parasitized Mean Across Bare and Mulch Treatments

There is no significant difference between the parasitized mean across bare and mulch treatments

Figure 10. Comparison Between Apterous Mean Across Bare and Mulch Treatments

There is no significant difference between the mean number of apterous across bare and mulch treatments

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Figure 11. Comparison Between Crown Weight Across Bare and Mulch Treatments

There is no significant difference between the average crown weight across bare and mulch treatments

Figure 12. Comparison Between Average Root Weight Across Bare and Mulch Treatments

There is no significant difference between the average root weight across bare and mulch treatments

Figure 13. Comparison Between Average Plant Weight Across Bare and Mulch Treatments

There is no significant difference between the average plant weight across bare and mulch treatments.

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Figure 14. Comparison Between Average Plant Height Across Buckwheat, Fava, Monoculture, and Mustard Treatments

Figure 15. Comparison of Presence of Apterous and Alate Aphids Between Buckwheat, Fava, Monoculture, and Mustard Treatments

Figure 16. Comparison of Average Number of

Leaves Across Buckwheat, Fava, Monoculture, and Mustard Treatments.

There is a statistically significant difference in the average number of leaves between all the intercrops.

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