Injury Caused by Western Tarnished Plant Bug (Hemiptera: Miridae) on Broccoli and Cauliflower in Laboratory Assays

Abstract

The polyphagous Lygus hesperus Knight is a serious pest on many crops in the western USA, including California’s central coast. Although L. hesperus adults can cause damage to broccoli and cauliflower, symptoms from their interactions with these plants are not fully characterized. Characterizing the feeding and ovipositional damage will help in the early diagnosis of the problem in the field and in greenhouses. Thus, the objective of this study was to characterize the feeding and ovipositional injury symptoms in broccoli and cauliflower after exposing 0, 1, 3, 5, and 10 adult L. hesperus to seedlings of broccoli and cauliflower for 24 h, 48 h, and 7 d. Although distorted and “blind” shoots were observed, feeding injury did not rapidly manifest into damage after 7 d post-exposure with high counts of adults on broccoli and cauliflower seedlings. The ovipositional injury was expressed as lesions that developed rapidly with a high density of adults in 24 h. The same levels of damage were observed with three or five adults to these hosts in 48 h. Significant positive correlations between the total eggs and lesions developed were observed on broccoli and cauliflower seedlings. After adult L. hesperus exposure, the growth of broccoli seedlings was reduced, but there was no effect on the growth of cauliflower seedlings. For diagnosis, data show that lesions are associated with adult L. hesperus ovipositional activity on these crops, which recommends thorough scouting and immediate application of plant protectants to reduce potential crop loss in greenhouses and in the field.

1. Introduction

The western tarnished plant bug, Lygus hesperus Knight (Hemiptera: Miridae), is an important pest on many crops [1,2], including brassicas [3], in the western USA, including central coast of California. Adult L. hesperus is sporadically reported in Brassica fields, such as broccoli (Brassica oleracea var. italica Plenck), cauliflower (B. oleracea L. var. botrytis), and Brussels sprouts (B. oleracea L. var. gemmifera), in the central coast of California (S.V.J. Pers. Observation). Previously, adult L. hesperus feeding has been reported on turnip (B. rapa var. rapa L.), radish (Raphanus sativus L.), and rape (Brassica napus L.) [3]. More than 57,588.8 ha of brassicas, mainly broccoli and cauliflower, are grown in California’s central coast, with an estimated value of >USD 957.6 million [4,5,6,7,8]. Although L. hesperus is reported in Brassica fields, the damage caused by feeding and oviposition injuries has not been systematically documented.

Lygus hesperus is a polyphagous pest [2]. The major crops affected are strawberry (Fragaria × ananasa Duchesne), onion (Allium cepa L.), clover (Trifolium spp.), alfalfa (Medicago sativa L.), lentils (Lens culinaris Medik.), pea (Pisum sativum L.), bean (Phaseolus vulgaris L.), dill (Anethum graveolens L.), carrot (Daucus carota L.) [3], celery [Apium graveolens dulce (Mill.) Pers.] [9,10,11], lettuce (Lactuca sativa L.) [12], and cotton (Gossypium hirsutum L.) [13]. In the central coast of California, L. hesperus are found on flowers of various weed species, such as wild radish (Raphanus raphanistrum L.), common groundsel (Senecio vulgaris L.), lupines (Lupinus spp.), milk thistle [Silybum marianum (L.) Gaertn.], and mustards (Brassica spp.), around production fields [2]. When the flowers senesce or stop producing flowers, adults move from these weeds to production fields, such as strawberries [2]. Adults and nymphs of L. hesperus probe host tissue using piercing–sucking mouthparts and pre-orally digest plant fluids and consume them [14,15,16]. In addition, females of L. hesperus oviposit pale, off-white bean-shaped eggs into plant tissue. The point of egg insertion gradually develops into lesions or streaks as the tissue surrounding the egg insertion continues to expand [11,12]. These lesions can cause economic damage if widespread and severe [11,12]. Moreover, the combined effects of feeding and oviposition injuries can result in seedling mortality in celery [11] and lettuce [12]. Thus, this study was designed to systematically characterize the damage from adult L. hesperus. However, the effects of adult L. hesperus injuries on broccoli and cauliflower seedlings have not been systematically characterized. In addition, from a management standpoint in the field and in greenhouses, it is important to characterize the symptoms associated with interaction between adult L. hesperus, and seedlings of broccoli and cauliflower. Broccoli plant samples were submitted to laboratory diagnosis, mostly seedlings with lesions and aborted terminal shoots. These Brassica plant samples could not be positively identified as having L. hesperus-induced damage without verifying how L. hesperus adults or nymphs interact with the tissue. Thus, the objective of this study was to characterize the damage symptoms produced by adult L. hesperus through feeding and oviposition on broccoli and cauliflower seedlings.

2. Materials and Methods

2.1. Plant and Insect

In 2016, trials were conducted in a greenhouse at the University of California Cooperative Extension (UCCE) Monterey County in Salinas, CA, USA. Broccoli ‘Heritage’ and cauliflower ‘Casper’ were the two hosts used in the trials. Broccoli seeds were purchased, whereas cauliflower seedling trays were obtained from a greenhouse facility in King City, CA, USA. In the central coast of California, broccoli seeds are typically direct-seeded, whereas cauliflower seedlings are transplanted in the field. Thus, broccoli seeds and cauliflower seedlings were planted in 3.78 L black plastic containers with a growing media (Sun Gro, Sunshine aggregate plus mix 4, Agawam, MA, USA). Broccoli seedlings were grown for five weeks in containers and used for the trial. The cauliflower seedlings were five weeks old when obtained from the greenhouse. They were retained for a week in the greenhouse for acclimatization and used in the trial. Seedling containers were maintained on greenhouse benches (Figure 1A) with irrigation at 2 d intervals for the duration of the trials. Fertilizer was not applied to the growing media, as the seedlings were only maintained for seven weeks in the greenhouse.

Adults of L. hesperus were collected in the field using sweep netting from Brassica weeds growing on the edges of production fields, such as strawberry, Brassica, and leafy greens in Salinas and Chualar, CA, USA. The collected adults were transported to the entomology laboratory at UCCE in a collapsible mesh cage (catalog#1466AV, Bioquip, Rancho Dominguez, CA, USA). Adults were sorted and temporarily maintained in the laboratory bench in the same mesh cages at ~22 °C and ~55% relative humidity (RH). The adults were used in the trials within 24 h.

2.2. Cage Design and Experiment Design

The trials were conducted using cylindrical cages (Figure 1A). The details of how the cages were prepared are described in Joseph et al. (2016) [11]. A 29.5 × 22.5 cm rectangular section of clear film (Grafix, Maple Heights, OH, USA) was rolled lengthwise, and the overlapping edge was taped. Using a glue gun, a no-see-um mesh fabric (cat# 7250NSW, Rancho Dominguez, CA, USA) was attached to one end of the cylinder. The other “hollow” end was buried 2.5 cm deep in the growing medium of a container (Figure 1A,B). Various counts of adult L. hesperus were caged with broccoli or cauliflower seedings (Figure 1B,C).

The treatments consisted of two factors: (1) counts of adult L. hesperus and (2) duration of exposure to host seedlings. The counts of adult L. hesperus were at five levels, i.e., 0, 1, 3, 5, and 10 adults per container, and the other factor, duration of exposure, was at three levels for 24 h, 48 h, and 7 d (Figure 1). The adult densities and duration of exposure provided low- and high-intensity scenarios, as determined by Joseph et al. (2016) [11] and Joseph (2019) [12]. The male–female ratio was approximately 50:50 in the natural field population (Joseph et al., 2016) [11]. Adults of L. hesperus were randomly collected from the maintenance cages and introduced to the broccoli and cauliflower seedling cages. The gender of adult L. hesperus was not determined to minimize any unintended handling injury to adults before introducing them to cages. The treatments were arranged in a completely randomized design with 10 replications on the greenhouse bench (Figure 1A). An individually caged plant was the experimental unit. After the respective exposure intervals (24 h, 48 h, and 7 d), the seedlings were extracted from the growing media and stored in plastic bags for evaluation. The seedlings were evaluated within 12 h after harvest.

2.3. Evaluation

Any evidence of feeding damage on the plant tissue, such as distortion or deformation at the growing center, was recorded (Figure 2A–C). In cauliflower, plants sometimes showed “blind” shoots where the growing point was destroyed. Females of L. hesperus oviposited singly near each other or far apart on plant tissue [11,12]. Over time, the tissue surrounding the point of egg insertion developed into a lesion (Figure 2D–F), as observed on celery and lettuce [11,12]. For evaluation, the number of lesions and L. hesperus eggs on leaves, petiole, and stem was examined and quantified under a dissecting microscope at 10× magnification. Because the opercula of the eggs are projected out of the epidermal layer, they are visible under 10× magnification. Over time, lesions expanded as the plant tissue increased, and the inserted eggs sometimes fell off. Most adults introduced inside cages were recovered. Similarly, feeding and oviposition could affect the normal growth of seedlings. Thus, the fresh weight and length of shoots were documented from the broccoli and cauliflower seedlings at 7 d post-exposure. For consistency, the seedlings were severed at the soil level, and shoots were individually weighed and measured using a balance (Ohus^®, Parsippany, NJ, USA) and a ruler, respectively.

2.4. Statistical Analyses

All statistical analyses were performed using SAS [17]. Because fewer feeding damage symptoms were observed, they were not analyzed. To determine the effects of adult L. hesperus, the number of L. hesperus eggs and discrete lesions was analyzed by L. hesperus count, exposure time, and their interaction. The data were subjected to a two-way analysis of variance (ANOVA) with interaction using the generalized linear model (PROC GLIMMIX) procedure in SAS. A value of one was added to every data point to satisfy the convergence criteria. The method used was “Laplace” with log-link in a Poisson distribution. Adult L. hesperus count, exposure time, and their interaction were the fixed effect, and replication was the random effect in the model. Means were separated using the Tukey–Krammer test (α = 0.05). Pearson’s correlation analysis was conducted between total L. hesperus eggs and lesions developed on the broccoli and cauliflower seedlings using the PROC CORR procedure in SAS.

The fresh weight and length of shoots at 7 d post-exposure were subjected to one-way ANOVA by adult L. hesperus count treatment using the generalized linear model (PROC GLIMMIX) procedure in SAS. The method used was “Laplace” with log-link in a Poisson distribution. Adult L. hesperus count was the fixed effect, and replication was the random effect in the model. Means were separated using the Tukey–Krammer test (α = 0.05).

Means and standard errors were calculated using the PROC MEANS in SAS.

3. Results

3.1. Feeding Injury

At 7 d post-exposure, the feeding damage observed was low, inconsistent, and nondetectable across the seedlings exposed to various L. hesperus densities. Only 1.3% of the 150 cauliflower seedlings in the study elicited “blind” shoot symptoms. Some broccoli seedlings elicited deformed shoots (Figure 2A–C), which were again only on ~1% of the broccoli plants at 7 d post-exposure. They were not statistically analyzed because of the low and inconsistent incidence of feeding damage symptoms.

3.2. Ovipositional Injury

After varied exposure periods, eggs were observed in the broccoli and cauliflower tissues in the midrib, leaf blade, and stem. As the plant tissue expanded, lesions developed where females inserted eggs, mostly on the midrib of broccoli and cauliflower leaves (Figure 2D–F). The eggs were often missing in some lesions.

For broccoli and cauliflower, the two-way ANOVA indicated that the numbers of eggs and lesions were significantly affected by L. hesperus densities, exposure time, and their interaction (

Table 1. The analysis of variance of the numbers of Lygus hesperus eggs and discrete lesions by adult L. hesperus densities (0, 1, 3, 5, and 10 individuals) exposed to broccoli and cauliflower seedlings for 24 h, 48 h, and 7 d in cages.

Treatment	Egg			Lesion
	F	df	p	F	df	p
Broccoli
L. hesperus	143.4	4126	<0.001	29.9	4126	<0.001
Exposure time	11.9	2126	<0.001	3.9	2126	0.023
L. hesperus × Exposure time	10.4	8126	<0.001	2.7	8126	0.010
Cauliflower
L. hesperus	77.5	4117	<0.001	25.2	4126	<0.001
Exposure time	23.7	2117	<0.001	4.2	2126	0.018
L. hesperus × Exposure time	28.7	7117	<0.001	2.4	8126	0.017

). Because the interaction effects differed significantly, a one-way ANOVA was conducted in terms of exposure time on L. hesperus densities.

3.2.1. Broccoli

The number of eggs was significantly different in the following order of adult treatments: 10 > 5 > 3 > 1 > nontreated control treatment after 24 h of adult exposure (F = 60.9; df = 4, 36; p < 0.001; Figure 3A). After 48 h of exposure, the number of eggs was significantly greater for the 10- and 5-adult treatments than for the 3-adult treatment followed by the 1- and 0-adult treatments (F = 56.5; df = 4, 36; p < 0.001). After 7 d of exposure, the significantly different order of egg counts was as follows: 10- = 5- > 3- > 1- > 0-adult treatments (F = 37.3; df = 4, 36; p < 0.001; Figure 3A).

After 24 h of exposure, the number of discrete lesions was significantly greater in the 10-adult treatment than in the 5-, 3-, 1-, and 0-adult treatments in broccoli (F = 13.5; df = 4, 36; p < 0.001; Figure 4A). At 48 h post-exposure, the pattern of lesion development was similar to that of the 24 h exposure, where significantly a greater number of lesions was observed in the 10-adult treatment than in the 5-, 3-, 1-, and 0-adult treatments (F = 13.4; df = 4, 36; p < 0.001). After 7 d of exposure, the number of lesions was significantly greater in the 10-adult treatment than in the 1- or 0-adult treatments (F = 8.1; df = 4, 36; p < 0.001; Figure 4A). There was no significant difference in the number of lesions between the 10-, 5-, and 3-adult treatments. Pearson’s correlation showed a significantly positive association between the total numbers of L. hesperus eggs and lesions developed (r = 0.789; N = 150; p < 0.001).

After 7 d post-exposure, the fresh weight of shoots (F = 15.9; df = 4, 36; p < 0.001; Figure 5A) and shoot lengths (F = 21.8; df = 4, 36; p < 0.001; Figure 5B) were significantly lower in the 10-adult treatment than in the 3- and 5-adult treatments followed by the 0- and 1-adult treatments.

3.2.2. Cauliflower

The egg densities were significantly greater in the 5-adult treatment than in the 10- and 1-adult treatments followed by the 0-adult treatment after 24 h of exposure (F = 19.8; df = 4,36; p < 0.001; Figure 3B). At 48 h post-exposure, the number of eggs was significantly greater in the 10-adult treatment than in the remaining adult treatments (F = 68.6; df = 4,36; p < 0.001). After 7 d of exposure, a significantly greater number of eggs was observed in the 10-adult treatment than in the 5- and 3-adult treatments, followed by the 1- and 0-adult treatments (F = 77.2; df = 3,27; p < 0.001; Figure 3B).

The number of lesions was significantly greater in the 10- and 5-adult treatments than in the 0-adult treatment after 24 h of exposure (F = 3.8; df = 4,36; p = 0.011; Figure 4B). At 48 h post-exposure, a significantly greater number of lesions was observed in the 10-adult treatment than in the 1-adult treatment followed by the 0-adult treatment (F = 8.7; df = 4,36; p < 0.001). After 7 d of exposure, the number of lesions was significantly greater in the 10-, 5-, and 3-adult treatments than in the 1- and 0-adult treatments (F = 15.4; df = 4,36; p < 0.001; Figure 4B). Pearson’s correlation showed a significantly positive association between the total numbers of L. hesperus eggs and lesions developed (r = 0.379; N = 140; p < 0.001).

After 7 d post-exposure, the fresh weight of shoots (F = 0.9; df = 4, 36; p = 0.464; Figure 5C) and shoot lengths (F = 0.2; df = 4, 36; p = 0.917; Figure 5D) were not significantly different between the adult density treatments.

4. Discussion

The results showed that the adult L. hesperus feeding did not rapidly induce damage to the broccoli and cauliflower seedlings. Although some broccoli and cauliflower seedlings developed distorted shoots and “blind” growing points, these damage symptoms were not widespread on these host plants at the seedling stage after adult exposure. This suggests that adult L. hesperus feeding damage may not widely develop within a short time on broccoli and cauliflower seedlings. This result is consistent with a previous study on lettuce, where adult L. hesperus feeding damage was not apparent within a short exposure period [12]. Conversely, feeding injury from adult L. hesperus caused dead necrotic tissue at the crown area of celery seedlings [11]. Thus, the prevalence of feeding injury varies among plant species. It is documented that L. hesperus prefers highly nutrient-dense plant parts, such as seeds [18] and developing embryos [2,19]. The incidence, frequency, and severity of adult L. hesperus feeding on various species of plants is still unclear. More studies are needed to understand the feeding patterns, such as preference and frequency, as well as the mechanisms leading to injury expression in these crops. Similarly, it is important to determine the effects of intraspecific competition among various densities of adult L. hesperus, which could have impacted the feeding and oviposition behavior.

Ovipositional injury was rapidly expressed as lesions on the broccoli and cauliflower seedlings with 10 adults in 24 h. Moreover, the same number of lesions that appeared with ten adults in 24 h was observed with five or three adults in 48 h. This suggests that, unlike feeding damage, ovipositional injury can cause noticeable damage to broccoli and cauliflower seedlings. Previously, a similar, rapid development of lesion damage was observed with a high density of adult L. hesperus in celery [11] and lettuce [12]. However, the economic impact of the injury caused by oviposition on broccoli and cauliflower in the field has not been completely studied. More eggs were observed in beans at peak flowering stages than in other stages of plant development [20]. Broccoli seeds are heavily sown on raised beds in two rows, and after seed germination, they are thinned at 15.2 cm spacing within two weeks. If L. hesperus adult attacks occur before thinning, the impact of ovipositional injury could be minimal, as most affected seedlings are most likely to be removed through thinning operation. However, if the adult L. hesperus attack occurs after thinning, it could cause crop losses by creating asynchrony in plant development and flower emergence and development in the field. Asynchronous plant development is a concern to growers as it causes a delayed and asynchronous emergence of florets, forcing multiple unsustainable harvest passes and leading to crop losses (S.V.J., unpublished data). With heavy lesion injury on midribs, the affected leaves develop yellowing and eventually drop off early (S.V.J. Per. Observation). The data showed that heavily infested seedlings developed more slowly than lightly infested broccoli. This suggests that the normal growth and development of young plants will be affected, and the damage related to oviposition will be expressed within 7 d of adult L. hesperus exposure. Thus, more research is warranted to understand the long-term effects of early infestation of adult L. hesperus, subsequent feeding, and ovipositional injury on broccoli leading up to harvest.

The number of eggs found in cauliflower plant tissue was relatively lower, with 10 adult L. hesperus exposed to seedlings in 24 h compared to broccoli (Figure 3). However, the development of lesions on the cauliflower was similar to that of the broccoli at 48 h or beyond. Compared to broccoli, cauliflower may be less preferred by adult L. hesperus as an ovipositional host. When given a choice of host plants, adult L. hesperus preferred foxtail barley, Hordeum jubatum L., and common groundsel, Senecio vulgaris L., for oviposition over alfalfa, Medicago sativa L., and other host species [21]. Although a choice test between broccoli and cauliflower plants was not conducted in the current study, a relatively lower number of eggs on the cauliflower compared to the broccoli after short exposure intervals suggests that cauliflower was preferred over broccoli by L. hesperus females for oviposition. In addition, some eggs likely dropped off from the tissue when the leaves expanded, which caused the injury, and the damage manifested over time on cauliflower.

There were no differences in plant weight and length of shoots for cauliflower regardless of adult L. hesperus density, but these parameters decreased progressively with an increase in adult L. hesperus density for broccoli. Although the exact reason is unclear, it is possible that the growth rate of cauliflower was affected by the conditions provided in the current study. Growing cauliflower is challenging compared to broccoli, as it is sensitive to variations in temperature, relative humidity, and moisture conditions in the soil [22]. For commercial production, cauliflower seeds are planted in trays in greenhouses, and the seedlings are raised for up to six weeks before being transplanted to the field [23]. Perhaps this high sensitivity of cauliflower to environmental conditions influenced reduced seedling growth, and the effects of adult L. hesperus interactions were not manifested.

In cauliflower, the plants exposed to five L. hesperus adults had more eggs than those exposed to ten adults. There is a chance that L. hesperus females oviposited multiple eggs in a relatively close spacing, and as the tissue expanded, some eggs dropped off. Joseph et al. (2016) observed multiple eggs oviposited by L. hesperus females in a tight spacing. Suppose cauliflower plants are vulnerable to adult L. hesperus populations. In that case, the seedlings must be protected using effective insecticides, such as neonicotinoids or pyrethroids, for a few weeks until they are fully established. Transplanted cauliflower seedlings will likely emit stress cues that could attract insect pests, including adult L. hesperus developing on flowering weeds in the surrounding landscape.

Based on the results, it appears that plant samples with aborted shoots and lesion symptoms from the field were more likely caused by L. hesperus feeding or ovipositional injury. Adult L. hesperus feeding injury symptoms did not rapidly develop in the broccoli and cauliflower seedlings. Ovipositional injury produces lesions on the midrib, as it can severely delay seedling development, especially for broccoli, and it could have serious adverse effects on yields. Thus, more research is needed to understand the long-term impact of feeding and ovipositional injury of adult L. hesperus during the early stages of seedling development. For diagnostic purposes, lesions on midribs and/or distorted and “blind” shoots imply possible adult L. hesperus ovipositional injury. This diagnosis will trigger the need for extensive scouting for adult L. hesperus in the affected areas of fields or greenhouses where samples are drawn. If the detection is positive, immediate follow-up should be carried out by applying plant protectants to reduce damage. This is the first study that has systematically characterized the feeding and oviposition injury of adult L. hesperus on broccoli and cauliflower.