Impact of potato psyllid (Hemiptera: Triozidae) feeding on free amino acid composition in potato

Abstract The impacts of potato psyllid (Bactericera cockerelli) feeding on potato foliage on the free amino acids (FAAs) composition in potato leaf and tubers were determined under greenhouse conditions. The free amino acids in plant extracts were separated by high‐performance liquid chromatography, and in both leaf and tuber samples, at least 17 FAAs were detected. Psyllid feeding significantly changed the levels of several FAAs in both leaf and tuber samples. The concentration of leucine increased 1.5‐fold, whereas that of serine and proline increased 2‐ and 3‐fold, respectively. In contrast, the concentrations of glutamic acid, aspartic acid and lyscine were significantly reduced by 42.0%, 52.1% and 27.5%, respectively. There were also significant changes in the levels of FAAs in the Zebra chip (ZC) infected tubers compared with the healthy tubers, and the levels of six of the FAAs increased, and the levels of nine of the FAAs decreased. The results from this study indicate that potato psyllid causes major changes in free amino acid composition of plant tissues, and this change in plant metabolism may contribute to the plant stress as indicated by increased levels of proline in the leaves and hence promoting the development of plant diseases such as ZC disease.     

Different host plant species modifies the susceptibility of Bactericera cockerelli to the entomopathogenic fungus Beauveria bassiana

Bactericera cockerelli (Sulc.) is a serious pest of solanaceous crops and a vector of the plant pathogen Candidatus Liberibacter psyllaurous. Entomopathogenic fungi are the most important biological control alternatives for this pest. Host plant species, however, can modify the outcomes of insect–pathogen interactions. We conducted laboratory experiments to quantify the virulence of two isolates of the entomopathogenic fungus Beauveria bassiana (Bals. [Vuill.]), BB40 and BB42, against third instar B. cockerelli nymphs maintained on chilli pepper plants. Owing to the lack of difference in virulence against B. cockerelli nymphs on chilli pepper between the two B. bassiana isolates, only BB42 was used to: compare virulence against nymphs maintained on either chilli pepper, potato or tomato; and in vivo conidia production from nymphs maintained on different host plants. Virulence of the two B. bassiana isolates against B. cockerelli nymphs was similar. Bactericera cockerelli nymphs maintained on tomato were more susceptible to B. bassiana than nymphs maintained on potato or chilli peppers. Infected nymphs maintained on chilli peppers produced the greatest number of conidia followed by infected nymphs maintained on tomato and potato. Host plant affected the susceptibility of B. cockerelli to B. bassiana isolate BB42 and subsequent conidia production. The implications of our results for microbial control of B. cockerelli by B. bassiana are discussed.     

Entomopathogenic fungi (Hypocreales) for control of potato psyllid, Bactericera cockerelli (Šulc) (Hemiptera: Triozidae) in an area endemic for zebra chip disease of potato

Potato psyllid, Bactericera cockerelli, is a serious pest of potato and other solanaceous vegetables in the United States, Mexico, Central America, and New Zealand and is responsible for transmission of Candidatus Liberibacter solanacearum which causes a disease known as “zebra chip” (ZC). Entomopathogenic fungi could provide a viable component for an integrated pest management strategy for control of B. cockerelli and other potato pest insects. Three field trials of commercial formulations of Metarhizium anisopliae (F 52®, Novozymes Biologicals) and Isaria fumosorosea (Pfr 97®, Certis USA) and abamectin (Agri-Mek®, Syngenta, USA) were conducted in Weslaco, Texas. Rates are expressed in quantity of product delivered in 375–470 l of water/ha. F 52 applied at 0.51, 1.1, and 2.2 l/ha and Agri-Mek applied at 584 ml/ha produced reductions of B. cockerelli eggs and nymphs of 45%, 59%, 67%, and 63%, respectively. Only Agri-Mek significantly reduced plant damage. Pfr 97 at 1.1 kg/ha with and without 1% Trilogy® (neem oil, Certis, USA), and Agri-Mek at 584 ml/ha resulted in psyllid reductions of 78%, 76%, and 84%, respectively. Significantly decreased plant damage and ZC symptoms were observed for all treatments. Tuber yields for Pfr plus Trilogy and Agri-Mek were significantly higher than the control. F 52 applied at 1.1 and 2.2 l/ha and Pfr 97 at 1.1 and 2.2 kg/ha produced 62%, 62%, 66%, and 65% reduction, respectively. Tuber yield for both rates of Pfr and the high rate of F 52 were significantly higher than the control. All fungal treatments significantly reduced plant damage and ZC symptoms.     

RNA Interference towards the Potato Psyllid,Bactericera cockerelli,Is Induced in Plants Infected with Recombinant Tobacco mosaic virus (TMV)

The potato/tomato psyllid, Bactericera cockerelli (B. cockerelli), is an important plant pest and the vector of the phloem-limited bacterium Candidatus Liberibacter psyllaurous (solanacearum), which is associated with the zebra chip disease of potatoes. Previously, we reported induction of RNA interference effects in B. cockerelli via in vitro-prepared dsRNA/siRNAs after intrathoracic injection, and after feeding of artificial diets containing these effector RNAs. In order to deliver RNAi effectors via plant hosts and to rapidly identify effective target sequences in plant-feeding B. cockerelli, here we developed a plant virus vector-based in planta system for evaluating candidate sequences. We show that recombinant Tobacco mosaic virus (TMV) containing B. cockerelli sequences can efficiently infect and generate small interfering RNAs in tomato (Solanum lycopersicum), tomatillo (Physalis philadelphica) and tobacco (Nicotiana tabacum) plants, and more importantly delivery of interfering sequences via TMV induces RNAi effects, as measured by actin and V-ATPase mRNA reductions, in B. cockerelli feeding on these plants. RNAi effects were primarily detected in the B. cockerelli guts. In contrast to our results with TMV, recombinant Potato virus X (PVX) and Tobacco rattle virus (TRV) did not give robust infections in all plants and did not induce detectable RNAi effects in B. cockerelli. The greatest RNA interference effects were observed when B. cockerelli nymphs were allowed to feed on leaf discs collected from inoculated or lower expanded leaves from corresponding TMV-infected plants. Tomatillo plants infected with recombinant TMV containing B. cockerelli actin or V-ATPase sequences also showed phenotypic effects resulting in decreased B. cockerelli progeny production as compared to plants infected by recombinant TMV containing GFP. These results showed that RNAi effects can be achieved in plants against the phloem feeder, B. cockerelli, and the TMV-plant system will provide a faster and more convenient method for screening of suitable RNAi target sequences in planta.     

Drought stress and pathogen infection alter feeding behavior of a phytopathogen vector

The impact of drought stress on tripartite plant-pathogen-vector interactions constitutes a complex and largely understudied field of plant-insect interaction. A number of studies explored these topics using aphid vectors of plant pathogens, but few have considered the interactions between drought-stressed plants and pathogen-transmitting psyllids. The potato psyllid, Bactericera cockerelli (Šulc) (Hemiptera: Triozidae), is one of the key pests of solanaceous crops in the USA that causes direct injury as well as indirect injury through transmission of a bacterial pathogen, Candidatus Liberibacter solanacearum (Lso), the causal agent of zebra chip. Previous studies explored the impact of Lso infection and drought stress on B. cockerelli development and reproductive rate separately, but no research to date has evaluated whether drought stress and Lso infection alter feeding behavior of the insects. We explored this using the electrical penetration graph (EPG) technique and monitored feeding behavior of Lso-infected and uninfected potato psyllids on well-watered and drought-stressed tomato (Solanum lycopersicum L., Solanaceae). We found that drought stress had a significant effect on feeding behavior associated with salivation into the phloem and phloem ingestion, both linked to Lso transmission. Furthermore, infected potato psyllids in particular produced a higher number of events associated with these feeding behaviors and remained in these phases longer in well-watered plants than in plants that were under drought stress. We also reported a new and previously undescribed waveform H of unknown biological function that was produced by the psyllids. This is the first study that considered the impact of bacterial infection and concomitant drought stress on feeding behavior of an insect quantified using EPG.     

Diversity of Endosymbionts in the Potato Psyllid, <Emphasis Type="Italic">Bactericera cockerelli</Emphasis> (Hemiptera: Triozidae), Vector of Zebra Chip Disease of Potato

Zebra chip disease is an emerging, serious disease of solanaceous crops and the causal agent is a bacterium “Candidatus Liberibacter solanacearum” (CLs), also known as “Candidatus Liberibacter psyllaurous”, which is transmitted by the potato psyllid, Bactericera cockerelli (Šulc). We performed bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) of the 16S rDNA genes to determine the bacterial microbiota in adult insects from CLs-uninfected and CLs-infected strains of B. cockerelli and potato leaf samples. We obtained sequences from five bacterial species among the two psyllid strains, including “Candidatus Carsonella ruddii”, Wolbachia, CLs, and two transient bacteria, Acinetobacter and Methylibium. We did not detect any common bacteria between psyllids and potato leaf samples using pyrosequencing. We performed PCR analysis using species-specific 16S rDNA primers to confirm pyrosequencing results in individual psyllids including eggs, early-instars, late-instars, and adults of both sexes from both CLs-uninfected and CLs-infected psyllid strains. The primary endosymbiont, “Candidatus Carsonella ruddii” and Wolbachia were detected in all life-stages and sexes of both strains using PCR analyses. The percentage of CLs-infected individuals increased from early-instar (0%), late-instar (40%) until adulthood (60%) in the CLs-infected strain. We believe that CLs levels in early-instars are probably too low to be detected by standard PCR. Using PCR analyses, we confirmed the presence of Acinetobacter in CLs-uninfected and CLs-infected adults (75 and 25%, respectively) but not Methylibium. Further, we detected Acinetobacter in potato leaves using PCR indicating that the psyllids may have acquired this bacterium via feeding on the host plant.     

Farm‐scale dispersal of Bactericera cockerelli in potato crops measured using Bt mark‐capture techniques

Natural populations of Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), also known as tomato/potato psyllid, were marked in potato [Solanum tuberosum L. (Solanaceae)] crops using Bacillus thuringiensis Berliner (Bt) to investigate the impact of dispersal on crop infestation and management of potential insecticide resistance in New Zealand. The technique was adapted from previous studies that used conventional spray applications of Bt to mark Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae), and identified marked individuals with selective microbiological assays and identification of characteristic crystal inclusions. Initially, marking rates of B. cockerelli were improved by using ultra‐low volume applications of undiluted Bt, but this result was not consistent. Several other pests and natural enemies were also marked. In mark‐capture studies, marked B. cockerelli were captured over 3 days on yellow sticky traps in small trap plots of potatoes at 60, 120, 180, 250, and 350 m from the sprayed crop. Bactericera cockerelli flight activity occurred throughout daylight hours with evidence of bimodal diurnal peaks. Significantly greater numbers of B. cockerelli were captured in downwind traps. The combined dispersal curve derived from two mark‐capture experiments estimated a mean dispersal distance for B. cockerelli of 100 m in 3 days and indicated that 10% of the population dispersed further than ca. 250 m. Over the period of a growing season, this level of dispersal suggests that B. cockerelli can disperse throughout a vegetable‐growing region, with implications for crop infestation and management of potential insecticide resistance.     

Insecticidal activity of entomopathogenic fungi (Hypocreales) for potato psyllid,Bactericera cockerelli (Hemiptera: Triozidae): Development of bioassay techniques,effect of fungal species and stage of the psyllid

The potato psyllid, Bactericera cockerelli (?ulc), is a pest of potato, tomato, and some other solanaceous vegetables and has also been incriminated in the transmission of a bacterial pathogen, Candidatus Liberibacter solanacearum, resulting in a serious disease known as ‘zebra chip’. Although there are several reports of fungal pathogens in psyllids, there are none from B. cockerelli, nor have any fungi been evaluated against it. Five isolates of fungi, one Beauveria bassiana, two Metarhizium anisopliae and two Isaria fumosorosea, were bioassayed against B. cockerelli on potato leaves under ideal conditions for the fungi. All applications were made with a Potter spray tower. With the exception of concentration-effect studies, all other applications were made using 10⁷ conidia/mL in a 2-mL aqueous suspension. All isolates except B. bassiana, produced 95–99% mortality, corrected for control mortality, in adults 2–3 days after application of conidia and 91–99% in nymphs 4 days after application. The corrected mortalities for adults and nymphs treated with B. bassiana were 53 and 78%, respectively, 4 days after application. I. fumosorosea Pfr 97 produced 95% corrected mortality in both first and late third instar nymphs. M. anisopliae (F 52) produced 96% corrected mortality in first and third instar nymphs. Pfr 97 and F 52 were evaluated for insecticidal activity against third instar B. cockerelli using 10⁵, 10⁶, and 10⁷ conidia per mL. Mortality produced by I. fumosorosea Pfr 97 ranged from 83 to 97% and that of M. anisopliae F 52 was 88 to 95% at these concentrations.     

Feeding disruption of potato psyllid,Bactericera cockerelli,by imidacloprid as measured by electrical penetration graphs

The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), is a serious pest of potatoes that can cause yield loss by direct feeding and by transmitting a bacterial pathogen, Candidatus Liberibacter psyllaurous (also known as Candidatus L. solanacearum), which is associated with zebra chip disease of this crop. Current pest management practices rely on the use of insecticides for control of potato psyllid to lower disease incidences and increase yields. Imidacloprid is typically applied at potato planting, and it remains unknown if imidacloprid has any effect on potato psyllid feeding behavior. Thus, our specific objectives of this study were to determine and characterize the effects of imidacloprid treatment (0.11 ml l^?1) to potato plants on adult potato psyllid feeding behavior 1, 2, and 4 weeks post‐application. Electrical penetration graph (EPG) recordings of potato psyllid feeding revealed six EPG waveforms, which include non‐probing (NP), intercellular stylet penetration (C), initial contact with phloem tissue (D), salivation into phloem sieve elements (E1), phloem sap ingestion (E2), and ingestion of xylem sap (G). The number of NP events and the duration of individual NP events significantly increased on plants treated with imidacloprid compared with untreated controls. Potato psyllids exhibited significant decreases in the number of phloem salivation events on plants treated with imidacloprid. Waveform durations and waveform durations per event for E2 and G were significantly decreased for psyllids on plants treated with imidacloprid compared with untreated controls. These data suggest that the effective use of imidacloprid to reduce transmission of Ca. Liberibacter psyllaurous is related to the negative effects of imidacloprid on psyllid feeding.     

Vector transmission efficiency of liberibacter by Bactericera cockerelli (Hemiptera: Triozidae) in zebra chip potato disease: effects of psyllid life stage and inoculation access period

Successful transmission of plant pathogens by insects depends on the vector inoculation efficiency and how rapidly the insect can effectively transmit the pathogen to the host plant. The potato psyllid, Bactericera cockerelli (Sulc), has recently been found to transmit "Candidatus Liberibacter solanacearum," a bacterium associated with zebra chip (ZC), an emerging and economically important disease of potato in several parts of the world. Currently, little is known about the epidemiology of ZC and its vector's inoculation capabilities. Studies were conducted in the field and laboratory to 1) assess transmission efficiency of potato psyllid nymphs and adults; 2) determine whether psyllid inoculation access period affects ZC incidence, severity, and potato yield; and 3) determine how fast the psyllid can transmit liberibacter to potato, leading to ZC development. Results showed that adult potato psyllids were highly efficient vectors of liberibacter that causes ZC and that nymphs were less efficient than adults at transmitting this bacterium. It was also determined that inoculation access period had little influence on overall ZC disease incidence, severity, and resulting yield loss. Moreover, results showed that exposure of a plant to 20 adult potato psyllids for a period as short as 1 h resulted in ZC symptom development. Furthermore, it was shown that a single adult potato psyllid was capable of inoculating liberibacter to potato within a period as short as 6 h, thereby inducing development of ZC. This information will help in developing effective management strategies for this serious potato disease.     

Age at reproductive maturity and effect of age and time of day on sex attraction in the potato psyllid Bactericera cockerelli

Abstract The potato psyllid, Bactericera (= Paratrioza) cockerelli (?ulc) (Hemiptera: Triozidae), is a major pest of potato. Studies were conducted to determine the age at which both males and females reach reproductive maturity and the effect of age and time of day on sex attraction. Adult B. cockerelli reach reproductive maturity within 48 h post‐eclosion, with females being mature on the day of eclosion and males at 1 day post‐eclosion. Oviposition generally began 2 days after mating but was delayed when females mated within 2 days post‐eclosion. In laboratory olfactometer assays, the age of females used as odor sources and the age of males assayed to these odors did not affect sex attraction, with both younger (1–4‐day‐old) and older (8–10‐day‐old) males being attracted to females, regardless of female age (1–4‐day‐old or 8–10‐day‐old). Males assayed to live females at different times during the photophase (between 8:00 and 20:00 hours) were attracted to females between 11:00 and 17:00 hours, showing a temporal periodicity in sex attraction with B. cockerelli at least during the photophase.     

Potential inter-guild interactions to enhance biological control of Bactericera cockerelli on tomatoes: a laboratory and cage study

The tomato–potato psyllid (TPP) Bactericera cockerelli, is a serious pest of solanaceous crops. Some populations are becoming pesticide-resistant, increasing the need for alternatives such as biological control (BC). This approach may be improved by combining different species of BC agents. We conducted three separate experiments to test four BC agents, either alone or combined with others: (1) A laboratory assay to test the effect of buckwheat (Fagopyrum esculentum) and alyssum (Lobularia maritima) flowers on the longevity of females of the parasitic wasp Tamarixia triozae; (2) A no-choice laboratory assay to investigate the consumption of B. cockerelli life stages by the predatory bug Engytatus nicotianae; (3) A cage experiment in a greenhouse to assess four natural enemy species against B. cockerelli on tomatoes: these were the predators Cleobora mellyi, Amblydromalus limonicus, E. nicotianae, and T. triozae. Access to buckwheat flowers allowed female T. triozae to live for an average of 10.9 days compared to 2.1 days with alyssum and 1.4 day with water but did not improve the BC of B. cockerelli. Adult E. nicotianae preyed on all offered B. cockerelli stages. In experiment 3, combinations of T. triozae with A. limonicus or E. nicotianae were not significantly better than single natural enemy species, except for the reduction of nymphal populations when A. limonicus and T. triozae were combined. Although there were few significant reductions in numbers of TPP when using natural enemy species combinations, some species showed good potential when used alone. We suggest testing earlier release of combinations of natural enemy for evaluate its impact on TPP.

     

Effects of liberibacter-infective Bactericera cockerelli (Hemiptera: Triozidae) density on zebra chip potato disease incidence, potato yield, and tuber processing quality

In plant pathosystems involving insect vectors, disease spread, incidence, and severity often depend on the density of the vector population and its rate of infectivity with the disease pathogen. The potato psyllid, Bactericera cockerelli (Sulc), has recently been associated with zebra chip (ZC), an emerging and economically important disease of potato in the United States, Mexico, Central America, and New Zealand. "Candidatus Liberibacter solanacearum," a previously undescribed species of liberibacter has been linked to the disease and is transmitted to potato by B. cockerelli. Experiments were conducted under laboratory and field conditions to determine the impact of B. cockerelli density on ZC incidence, potato yield, and tuber processing quality. Insect densities ranging from one to 25 liberibacter-infective psyllids per plant were used during the experiments. Results showed that a single adult potato psyllid was capable of inoculating liberibacter to potato and causing ZC disease after a 72-h inoculation access period and was as damaging as 25 psyllids per plant. In addition, ZC-diseased plants showed a sharp reduction in tuber yield but the disease response was independent of the density of psyllids. Furthermore, both glucose and sucrose were found to have highly elevated concentrations in ZC-diseased potato tubers compared with noninfected ones and psyllid density did not vary the response. The high reducing sugar concentrations found in ZC-infected potato tubers are believed to be responsible for browning and reduced quality in processed ZC-infected tubers. This information could help ZC-affected potato producers in making effective management decisions for this serious disease.     

Zebra chip disease incidence on potato is influenced by timing of potato psyllid infestation, but not by the host plants on which they were reared

Abstract  The Zebra chip (ZC) syndrome is an emerging disease of potato and a major threat to the potato industry. The potato psyllid, Bactericerca cockerelli (Sulc) is believed to be a vector of the ZC pathogen, which is now thought to be Candidatus Liberibacter, a bacterium. To further understand the relationship between potato psyllid infestation and ZC disease expression, healthy potato plants at different growth stages (4, 6 and 10 weeks after germination) were exposed separately to potato psyllids that were separately reared on four solanaceous hosts plants (potato, tomato, eggplant or bell pepper) for more than 1 year. ZC symptoms, leaf rates and total nonstructural carbohydrate accumulation in leaves and tubers of healthy and psyllid-infested plants were monitored and recorded. Typical ZC symptoms were observed in leaves and tubers of all plants exposed to potato psyllids regardless of the host plant on which they were reared. This was also accompanied by significant reductions in net photosynthetic rate. Caged potato plants without exposure to potato psyllids (uninfested controls) did not show any ZC symptom in both foliage and in harvested tubers. Foliage damage and ZC expression were most severe in the potato plants that were exposed to potato psyllids 4 weeks after germination compared to plants infested at later growth stages. Tubers from potato psyllid-infested plants had significantly higher levels of reducing sugars (glucose) and lower levels of starch than those in healthy plants, indicating that potato psyllid infestation interfered with carbohydrate metabolism in either leaves or tubers, resulting in ZC expression.     

Horizontal Transmission of "Candidatus Liberibacter solanacearum" by Bactericera cockerelli (Hemiptera: Triozidae) on Convolvulus and Ipomoea (Solanales: Convolvulaceae)

“Candidatus Liberibacter solanacearum” (Proteobacteria) is an important pathogen of solanaceous crops (Solanales: Solanaceae) in North America and New Zealand, and is the putative causal agent of zebra chip disease of potato. This phloem-limited pathogen is transmitted to potato and other solanaceous plants by the potato psyllid, Bactericera cockerelli (Hemiptera: Triozidae). While some plants in the Convolvulaceae (Solanales) are also known hosts for B. cockerelli, previous efforts to detect Liberibacter in Convolvulaceae have been unsuccessful. Moreover, studies to determine whether Liberibacter can be acquired from these plants by B. cockerelli are lacking. The goal of this study was to determine whether horizontal transmission of Liberibacter occurs among potato psyllids on two species of Convolvulaceae, sweet potato (Ipomoea batatas) and field bindweed (Convolvulus arvensis), which grows abundantly in potato growing regions of the United States. Results indicated that uninfected psyllids acquired Liberibacter from both I. batatas and C. arvensis if infected psyllids were present on plants concurrently with the uninfected psyllids. Uninfected psyllids did not acquire Liberibacter from plants if the infected psyllids were removed from the plants before the uninfected psyllids were allowed access. In contrast with previous reports, PCR did detect the presence of Liberibacter DNA in some plants. However, visible amplicons were faint and did not correspond with acquisition of the pathogen by uninfected psyllids. None of the plants exhibited disease symptoms. Results indicate that horizontal transmission of Liberibacter among potato psyllids can occur on Convolvulaceae, and that the association between Liberibacter and Convolvulaceae merits additional attention.     

Biology and epidemics of Candidatus Liberibacter species,psyllid‐transmitted plant‐pathogenic bacteria

Candidatus Liberibacter species are Gram‐negative bacteria that live as phloem‐limited obligate parasites in plants, and are associated with several plant diseases. These bacteria are transmitted by insects called psyllids, or jumping plant lice, which feed on plant phloem sap. Citrus huanglongbing (yellow shoot) or citrus greening disease is associated with three different species of Ca. Liberibacter – Ca. L. asiaticus, Ca. L. africanus and Ca. L. americanus – all originally found on different continents. Ca. L. asiaticus is the most severe pathogen, spread by Asian citrus psyllid Diaphorina citri and causing devastating epidemics in several countries. Ca. L. africanus occurs in Africa where it is spread by the African citrus psyllid Trioza erytreae. Ca. Liberibacter solanacearum is associated with diseases in several solanaceous plants, and transmitted by potato psyllid Bactericera cockerelli. Zebra chip disease is causing large damage in potato crops in North America. In Europe Ca. Liberibacter solanacearum is associated with diseases of the Apiaceae family of plants, carrot and celery, and transmitted by psyllids Trioza apicalis and Bactericera trigonica. When Ca. Liberibacter is suspected as the disease agent, the diagnosis is confirmed by DNA‐based detection methods. Ca. Liberibacter‐associated plant diseases can be controlled by using healthy plant propagation material, eradicating symptomatic plants, and by controlling the psyllid populations spreading the disease.     

Quenching autofluorescence in the alimentary canal tissues of Bactericera cockerelli (Hemiptera: Triozidae) for immunofluorescence labeling

Immunofluorescence has been widely used to localize microbes or specific molecules in insect tissues or cells. However, significant autofluorescence is frequently observed in tissues which can interfere with the fluorescent identification of target antigens, leading to inaccurate or even false positive fluorescent labeling. The alimentary canal of the potato psyllid, Bactericera cockerelli ?ulc, exhibits intense autofluorescence, hindering the application of immunolocalization for the detection and localization of the economically important pathogen transmitted by this insect, “Candidatus Liberibacter solanacearum” (Lso). In the present study, we tested the use of irradiation, hydrogen peroxide (H₂O₂) and Sudan black B (SBB) treatments to reduce the autofluorescence in the B. cockerelli alimentary canal tissues. Furthermore, we assessed the compatibility of the above‐mentioned treatments with Lso immunolocalization and actin staining using phalloidin. Our results showed that the autofluorescence in the alimentary canal was reduced by irradiation, H₂O₂, or SBB treatments. The compatibility assays indicated that irradiation and H₂O₂ treatment both greatly reduced the fluorescent signal associated with Lso and actin. However, the SBB incubation preserved those target signals, while efficiently eliminating autofluorescence in the psyllid alimentary canal. Therefore, herein we propose a robust method for reducing the autofluorescence in the B. cockerelli alimentary canal with SBB treatment, which may improve the use of immunofluorescence labeling in this organism. This method may also have a wide range of uses by reducing the autofluorescence in other arthropod species.