The cyanogenic potential is measured as the amount of pre-cyanogenic compounds contained in the plant tissue . The cyanogenic capacity is measured as the amount of cyanide released by a given quantity of plant tissue over a unit of time . The enzymatic activity, which can be variable across species and genotypes, will determine how closely measurements of cyanogenic potential and cyanogenic capacity correspond . The interaction between cyanogenic plants and insect herbivores is complex and maybe be affected by several biotic and abiotic factors . As with other chemical defense mechanisms, specialist insect herbivores may have adaptations that protect them from cyanide produced by their host plant including metabolizing it for use of the nitrogen in protein synthesis or sequestering it for their own defense . Generalist herbivores may avoid cyanide intoxication by balancing their diet with cyanogenic and acyanogenic foods .As a defense mechanism, cyanogenesis can operate in two ways depending on the cyanogenic capacity of a given plant tissue: deterrence or intoxication . Cyanogenesis is not a universally effective deterrent and for some insects cyanide may even act as a phagostimulant . Cyanogenesis seems most effective as a deterrent when the cyanogenic capacity is high, release is rapid, and the insect herbivore is an opportunistic generalist rather than a well-adapted specialist . Below a certain threshold, dependent on the herbivore, square pots cyanogenesis is ineffective as a deterrent . Intoxication by cyanide consumption typically occurs when herbivores consume large amounts of plant material with lower cyanogenic capacity . In this sort of situation, cyanide may be released within the digestive track, causing lethal damage or inhibiting growth of the insect .
Plants with higher cyanogenic capacity may be rejected before an insect can consume a sufficient dose. There is variation in the susceptibility of Lima bean to damage by L. hesperus but this has only been catalogued within a small number of commercial cultivars. The first step of this study is to catalogue this variation within a more diverse panel of accessions. From this research, the University of California Davis Dry Bean Breeding program will be able to select better parents and introduce more diversity into the Lima Bean breeding pipeline. In addition to characterizing the variation in L. hesperus tolerance or resistance, understanding more about the possible mechanisms that contribute to this phenotype will help target selection in breeding. To do this a field study in which multiple varieties were vacuum sampled will be analyzed to demonstrate the choice of L. hesperus in the field when multiple varieties are present. It is unknown if cyanogenesis is an effective defense againstL. hesperus in Lima bean. In combination, these studies illustrate the extent of variation in L. hesperus resistance or tolerance phenotypes in Lima bean as well as determine if cyanogenesis is induced by L. hesperus presence and if there is a negative correlated between cyanide and L. hesperus population growth. The other block was a control not treated with insecticides. In 2019, UC 92 plots were vacuumed to in each block to verify that L. hesperus levels were lower in the insecticide treated plots. In 2021, the vacuum equipment was unavailable, so water traps were used to verify the difference in L. hesperus pressure between blocks. Both fields were drip irrigated and conventionally managed. Each variety was planted in a single row in eight plots of 4.5 meters . The plots were randomized within eight sub-blocks. Field notes on days to flowering, growth habit, seed color, and flower color were taken. Yield and 100-seed-weight measurements were conducted after harvest. Four ofthe lines were excluded from the analysis due to poor germination or photoperiod sensitivity.
In a small trial at the University of California Davis Student Organic Farm, nine Lima bean varieties were planted on June 1, 2017, at 38°32’32.5″N 121°46’01.3″W . The field was flood irrigated and organically managed. Every variety was planted in two plots, one of which was randomly assigned a location within each of two blocks. Each plot was 20 feet long and six 30-inch rows wide. Starting at the time of flowering, July 21, 2017, the middle two rows of every plot were vacuumed each week between 11am and 1pm. Samples for each plot were bagged and then frozen. Insects were then transferred to vials of ethanol and adult L. hesperus in the sample were counted and sexed. Adult L. hesperus are highly mobile and can readily fly between small plots. Nymph counts would therefore have been a better measurement to take. However, nymphs were not counted because many were crushed by the force of the vacuum or were two small to be accurately identified with the available expertise. The middle two rows of each plot were harvested measured for total yield and 100 seed weight. The variety UC Lee was removed from the study due to poor germination rates.Five cultivars of California-adapted Lima bean were selected for this study with the aim of representing the diversity of seed size, growth habit, cyanogenic capacity, and tolerance of L. hesperus . All the varieties had white seed coats as this is the market standard for dry Lima beans produced in California. Prior research found that there is not a correlation between seed coat color and cyanogenesis . Originally, one wild accession was included for comparison but due to photoperiod sensitivity and delayed phenology, it proved infeasible to collect samples from these plants in synchrony with the others.In four greenhouse plantings, plants of each variety were individually germinated from seed in azalea pots with approximately two liters of UC Agronomy potting soil mix. Each pot was placed in its own cage with drip line for water and fertilizer . Each cage was randomly assigned a position in the greenhouse. Three flowers and three young podsof each plant were collected one, two, and three weeks after flowering. Flowers were selected with white petals, indicating that the day of sampling was their first to open. Immature pods were approximately 2cm long . Additionally, succulent mature seed tissue was collected four weeks after flowering were sampled. Mature seed samples consisted of a slice of the bean from the opposite side of the hilum from the micropyle of approximately 200mg that would fit into a 96-well plate collection tube . All samples were frozen at -80°C and later analyzed for cyanogenic capacity using the Feigl-Anger paper assay . While some have critiqued this method for being only semiquantitative, it was selected based on the available resources and practicality for analyzing large numbers of samples .Half of the plants were randomly assigned to a treatment group which had adult L. hesperus added to their bug dorm one week after flowering. In the pilot study, 25 adult L. hesperus were added. The resulting level of herbivory was high and, as a result, the susceptible varieties, UC 92 and UC Lee, had insufficient flowers survive for sampling or pod development. In the subsequent rounds of the experiment a total of only 14 adult L. hesperus were added, square plant pot seven one-week-old adult males and seven one-week-old adult females. This level of herbivory preserved sufficient flowers for sampling and pod development on susceptible plants. The one-week-old adult insects were added after the week one flower and pod samples were collected so that those samples had no interaction with the L. hesperus. All cages had a low level of thrips infestation; however, the greenhouses were not treated with insecticide during the study. All L. hesperus introduced to cages in the experiment were one-week post emergence adults reared in a colony founded by individuals collected from Lima bean and alfalfa fields in the fall of 2019 and 2020 . The colony was maintained at 20°C and 12 h of photo period. Adults were held in 30.5cm cube collapsible cage with a bedding of shredded white printer paper, a water-soaked organic cotton round , hulled sunflower seeds, and fresh organic green beans supplied three times a week. The egg-laid beans were moved to rearing tubs where they emerged in approximately 7-10 days. Nymphs were supplied with a bedding of shredded white printer paper and green beans three times per week and moved to adult cages at the time of emergence.The pilot study, September 2019-December 2020 was conducted in a small greenhouse at the Orchard Park Greenhouse Facility at UC Davis and included only two plants of each variety.
The second round conducted in December 2019-March 2020 was conducted in the same greenhouse. In round two, six plants of each variety were planted and divided equally between the two treatment groups. The third round, March 2020-May 2020, was conducted in a neighboring greenhouse that had been enclosed with black plastic drapes to exclude all-natural light. Artificial lights lit the greenhouse in a 12-hour photo period. This was done in an attempt to include a photo period sensitive wild Lima bean accession in the study but even with this treatment, it was not possible to synchronize its flowering with that of the commercial cultivars. In round three, six plants of each variety were planted and divided equally between the two treatment groups. In the fourth round of the study, January 2021-April 2021, the experiment was planted in a neighboring greenhouse with natural light. In round four, eight plants of each variety were planted and divided equally between the two treatment groups. In each round of the experiment, an equal number of plants for each variety and treatment group were planted. However, due to poor germination, not every plant survived to participate in the study. Across the three plantings there were a total of 16 replicates of each variety divided between two treatment groups – so eight plants per treatment. In all rounds of the studies, cages were randomly assigned positions within the greenhouse using the Microsoft Excel random number generator function. All the flower and pod samples were frozen at -80°C for several months before they were processed with a colorimetric assay with Feigl-Anger paper . Defrosting samples were exposed to Feigl-Anger paper for 0-15 minutes, 15-30 minutes, 30-60 minutes, and 60-90 minutes after being removed from the freezer. The results of the assay were scanned and analyzed using the readplate2 plugin on ImageJ 1.52q . This semiquantitative method provided a measurement of the intensity of blue produced by the interaction of volatilized HCN and the chemical treatment of the Feigl-Anger paper. Since the volatilized HCN had to be synthesized from enzymatic activity in thawing sample tissues, a standard of KCN in NaOH solution, used in other studies, was not considered sufficiently comparable to use in estimating the quantities of cyanide released from tissues during each exposure window.To understand how well L. hesperus survive and reproduce on varieties of Lima bean, populations founded by adult L. hesperus added to cages in the cyanogenic response experiment were collected and analyzed after three weeks. The development of L. hesperus is temperature dependent but takes approximately 4 weeks at 20°C, with this being the approximate temperature of the greenhouses in which this experiment was conducted . Three weeks is therefore not enough time for eggs to be laid and the resulting offspring to develop into adults and so the adults collected were survivors of the initial introduction rather than newly developed adults. The number of surviving adults and nymphs were counted for each cage in the treatment group. In rounds three and four, the nymphal instars were identified to indicate the speed of development for L. hesperus on the various varieties of Lima bean. Analysis All statistical analysis was conducted in R version 4.2.1 . For the final analysis, only the 60–90-minute exposure window was used due to concerns that plate position may have affected the results of earlier windows since samples on the outside of the 96-well plate may have defrosted more quickly than samples in the interior of the 96-well plate . This design flaw was not apparent in the time trial since a smaller number of samples were used and so the thermal mass of the plate was lower. It has been noted as an important lesson in experimental uniformity. Prior to publication, a statistical model including plate position may be tested to analyze results from earlier exposure windows.