Lytic enzymes induced by Pseudomonas fluorescens and other biocontrol organisms mediate defence against the anthracnose pathogen in mango

Talc-based bioformulations containing cells of Pseudomonas fluorescens, Bacillus subtilis and Saccharomyces cerevisiae were evaluated for their potential to attack the mango (Mangifera indica L.) anthracnose pathogen Colletotrichum gloeosporioides Penz. under endemic conditions. The preharvest aerial spray was given at fortnightly and monthly intervals. The plant growth-promoting rhizobacteria Pseudomonas fluorescens (FP7) amended with chitin sprayed at fortnightly intervals gave the maximum induction of flowering, a yield attribute in the preharvest stage, consequently reduced latent symptoms were recorded at the postharvest stage. An enormous induction of the defence-mediating lytic enzymes chitinase and β-1,3-glucanase was recorded in colorimetric assay and the expression of discrete bands in native PAGE analysis after FP7 + chitin treatment. The enhanced expression of defence-mediating enzymes may collectively contribute to suppress the anthracnose pathogen, leading to improved yield attributes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Secondary hosts of the Asian citrus psyllid,Diaphorina citri Kuwayama: Survivorship and preference

The Asian citrus psyllid, Diaphorina citri Kuwayama, is the most economically important and difficult to manage citrus pest in Florida due to its role as vector of huanglongbing or citrus greening disease. Spread of the disease is a function of dispersal patterns and thus influenced by availability of suitable hosts. Young leaves of citrus or related plants are required for reproduction, but in their absence, secondary hosts may provide needed resources to enhance survival. Therefore, survivorship on and preference for three abundant weed species was investigated. The suitability of potential secondary host plants Bidens alba (L.), DC, Ludwigia octovalvis (Jacqu.) P. H. Raven, and Eupatorium capillifolium (Lam.) Small was compared to a reproductive host, Murraya paniculata (L.) Jack, in no‐choice tests by assessing survivorship of D. citri adults confined to these plants in cages. Preference was evaluated by choice tests where D. citri adults were released into cages containing all three secondary hosts alone or with M. paniculata. Both B. alba and E. capillifolium increased D. citri survivorship by twofold compared to starvation conditions with only water available. Choice trials revealed no difference in initial selection between true and secondary hosts; however, the true host was favoured over time. This result suggested that hosts were selected initially by sight, and only later by taste and/or smell. While secondary hosts are unable to support reproduction or long‐term survival, these findings establish the ability of D. citri to use secondary hosts that are ubiquitous in Florida citrus groves as temporary reservoirs for food and moisture when ideal host conditions are scarce or absent.     

Biological control of the citrus red mite <Emphasis Type="Italic">Panonychus</Emphasis><Emphasis Type="Italic">citri</Emphasis> by the predator mite <Emphasis Type="Italic">Typhlodromus athiasae</Emphasis> on two citrus cultivars under greenhouse conditions

This study examined the efficacy of the predatory mite Typhlodromus athiasae Porath and Swirski (Acari: Phytoseiidae) as a biological control agent of the citrus red mite Panonychus citri (McGregor) (Acari: Tetranychidae) on seedlings of Washington and Valencia citrus cultivars at 1:10, 1:20 and 1:40 predator:prey release ratios under greenhouse conditions. At predator:prey ratios of 1:10, significant reductions on P. citri populations were observed one week after release of T. athiasae, and populations remained at low levels thereafter. The highest mean numbers of P. citri were found in the third week on the Washington cultivar and in the fourth week on Valencia, in a control group with no predators. This study demonstrates the potential of T. athiasae to effectively control P. citri on Washington and Valencia cultivars under greenhouse conditions at predator:prey ratios of 1:10. However, T. athiasae was unable to control the citrus red mite populations when the predator:prey ratio was reduced to 1:40. We therefore recommend a release ratio of 1:10 for effective control of P. citri in greenhouses on seedlings of Washington and Valencia citrus.     

Significance of Microsclerotia in the Epidemiology of Black Pepper Anthracnose and an Approach for Disease Management in Nurseries

Anthracnose incited by Colletotrichum gloeosporioides (Penz.) Penz. and Sacc. is a wide spread and economically important disease of black pepper. In the present study, role of microsclerotia (MS) in the trans‐seasonal perpetuation of C. gloeosporioides was investigated. Microscopical examination of the runner shoots exhibiting necrotic lesions revealed the presence of dark, melanized structures which resembled MS. The excised necrotic regions when subjected to high humidity produced acervulus with setae. Under in vitro conditions, C. gloeosporioides produced MS predominantly on the aerial surface as inseparable congregations, enmeshed in the mycelial mats in potato dextrose broth and as individual units 7–8 days after incubation on glass slides. Sequential events in the formation of MS included germination of conidia, formation of conidial anastomosis tubes, aggregation of hyphae, and the formation of melanized microsclerotial bodies. Three types of microsclerotial germination were observed under in vitro conditions viz., sporogenic, myceliogenic and both. PCR confirmation with CgInt species‐specific primer and ITS4 resulted in 450‐bp amplification. Since, runner shoots are predominantly used as propagating material in black pepper, an approach was devised to manage anthracnose under nursery conditions by treating the 2‐ to 3‐node cuttings (nursery planting material) with carbendazim (12%)—mancozeb (63%) @ 0.1% for 30 min. The results of the study suggests a new facet in the disease cycle of black pepper anthracnose, indicating that the pathogen survives as microsclerotia in planta and could act as a potential source of inoculum.     

A preliminary study on the culture ofDiscomycetes,especially the perfect stage

Summary The author made a study of the culture ofDiscomycetes. About fourty species were studied in vitro. An ascertaining of the conditions inducing the perfect stage was aimed at and as far as these fructifications developed they were studied. From six of these fungiviz., Cenangium ferruginosum Fr.,Encoelia fascicularis (Fr.) Karst.,Mollisia spec.,Pezicula cinnamomea (Fr.) Sacc.,Phialea acuum (Alb. en Schw. ex Fr.) Rehm andSclerotinia sclerotiorum (Lib.) de Bary, apothecia were obtained in culture, the others failed to do so.     

Production of Industrial Enzymes (Amylase,Carboxymethylcellulase and Protease) by Bacteria Isolated from Marine Sedentary Organisms

The abilities of bacteria isolated from eight marine sedentary organisms, six marine sponges (Spirastrella sp., Phyllospongia sp., Ircinia sp., Aaptos sp., Azorica sp. and Axinella sp.), one soft coral (Lobophytum sp.) and one alga (Sargassum sp.) to produce industrial enzymes (amylase, carboxymethylcellulase and protease) were examined. The mean total viable counts of the bacterial isolates ranged from 8.7 × 10⁴ to 8.4 × 10⁵ cfu/g wet weight of the organism. All eight organisms harboured amylase (0.05–0.5 IU/ml), carboxymethylcellulase (0.05–0.5 IU/ml) and protease (0.1–0.5 IU/ml) producing bacteria. Of 56 bacterial strains tested, as many as 60 to 83% of the strains produced at least one of the three enzymes, and 47% of strains were able to produce all three enzymes. High activities (> 0.5 IU/ml) of the three enzymes were recorded in bacterial strains belonging to the genera Alcaligenes and Bacillus. From the results of this study, it appears that bacteria associated with marine sedentary organisms are the novel source of industrial enzymes for possible commercial applications and may play an important role in enzyme‐catalysed organic matter cycling in marine environments.     

Effect of plant barriers and citrus leaf age on dispersal of Diaphorina citri (Hemiptera: Liviidae)

Studies designed to measure dispersal capacity of Diaphorina citri Kuwayama (Hemiptera: Liviidae) are needed to provide the epidemiological knowledge necessary to improve management of citrus huanglongbing. In this study, a mark–release–recapture technique was used to investigate whether 1) host or non‐host plants of D. citri can act as barriers for dispersing insects and 2) presence or absence of young citrus leaves influence movement of D. citri towards citrus plants. The experimental field consisted of four circular and adjacent areas containing citrus trees, Citrus sinensis (L.) Osbeck cv. ‘Hamlin’, planted in concentric circles at 18, 24 and 30 m from the release centre. Insect activity was monitored by recapturing at each distance using yellow stick traps. Dense plantings of tall non‐host plants of D. citri such as corn had no effect on insect dispersal towards citrus plants when compared to a shorter cover crop such as grass. In contrast, suitable host plants acted as traps decreasing movement of D. citri. Diaphorina citri dispersed at greater speeds in the absence of young leaves reaching 140 m within 6 hours after release, whereas in the presence of young leaves, individuals reached at most 60 m at 1 day after release. Results suggest that D. citri control measures may be more efficient during periods of highest vegetative activity when insects are less active. Moreover, the use of suitable host plants for D. citri as trap plants may be a potential tactic to prevent movement of insects into the crop.     

Pseudomonas protegens CS1 from the lemon phyllosphere as a candidate for citrus canker biocontrol agent

• Citrus canker is a worldwide‐distributed disease caused by Xanthomonas citri subsp. citri. One of the most used strategies to control the disease is centred on copper‐based compounds that cause environmental problems. Therefore, it is of interest to develop new strategies to manage the disease. Previously, we reported the ability of the siderophore pyochelin, produced by the opportunistic human pathogen Pseudomonas aeruginosa, to inhibit in vitro several bacterial species, including X. citri subsp. citri. The action mechanism, addressed with the model bacterium Escherichia coli, was connected to the generation of reactive oxygen species (ROS). This work aimed to find a non‐pathogenic strain from the lemon phyllosphere that would produce pyochelin and therefore serve in canker biocontrol.
• An isolate that retained its capacity to colonise the lemon phyllosphere and inhibit X. citri subsp. citri was selected and characterised as Pseudomonas protegens CS1. From a liquid culture of this strain, the active compound was purified and identified as the pyochelin enantiomer, enantio‐pyochelin.
• Using the producing strain and the pure compound, both in vitro and in vivo, we determined that the action mechanism of X. citri subsp. citri inhibition also involved the generation of ROS. Finally, the potential application of P. protegens CS1 was evaluated by spraying the bacterium in a model that mimics the natural X. citri subsp. citri infection.
• The ability of P. protegens CS1 to reduce canker formation makes this strain an interesting candidate as a biocontrol agent.

     

An assessment of interspecific competition between two introduced parasitoids of Diaphorina citri (Hemiptera: Liviidae) on caged citrus plants

Two parasitoids attacking nymphs of Asian citrus psyllid,Diaphorina citri Kuwayama (Hemiptera:Liviidae),Tamarixia radiata (Waterston)(Hymenoptera:Eulophi- dae)and Diaphoreneyrtus aligarhensis (Shafee,Alam &Agarwal)(Hymenoptera:Encyrtidae)are being released in California,USA in a classical biological control program. To evaluate the effect of multiple parasitoid species on D.citri mortality,we conducted mesocosm experiments under controlled conditions using a complete block design with 6 treatments (D.cirri nymphs exposed to:no parasitoids;D.aligarhensis or T.radiata alone;D.aligarhensis or T.radiata released first (by 48 h);and both species released simultaneously).Parasitism of D.citri nymphs by T.radiata exceeded 60%and was unchanged when D.aligarhensis were present.Parasitism by D.aligarhensis was greatest when T. radiata was absent (-28%)and was reduced in all treatments with T.radiata present (<3%).D.cirri mortality and parasitoid-related mortality olD.citri was consistent across parasitoid treatments.Laboratory results suggest that competition between D.aligarhensis and T.radiata is asymmetric and favors T.radiata.It may be difficult for D.aligarhensis to contribute significantly to D.cirri biological control where T.radiata is present.However, results reported here suggest that competition between T.radiata and D.aligarhensis is not likely to reduce parasitism by T.radiata or reduce parasitoid-induced mortality of D.citri.     

The process of population increase and patterns of resource utilization of two spider mites,Oligonychus ununguis (Jacobi) andPanonychus citri (McGregor), under experimental conditions (Acari: Tetranychidae)

Summary Pattern of population growth and characteristics of habitat utilization and of migration by two species of spider mites were studied under experimental conditions. The population growth ofOligonychus ununguis (Jacobi) on a chestnut occurred only on a single mite-release leaf over a long period, and few individuals moved away. Most of the 2nd progeny generation females of this species emigrated from the mite-release leaf as well as the sapling by means of ballooning threads. During this growth period, population density on the mite-release leaf levelled off, whereas that on the sapling increased. In contrast, the foundress ofPanonychus citri (McGregor) on citrus actively moved over several neighbouring leaves, and until the 2nd progeny generation females emerged, individuals were distributed over all the sapling leaves by means of walking. Emigration from the sapling was not observed until the 2nd progeny females emerged, and after that the mites emigrated by means of ballooning threads. The population density ofP. citri on the sapling levelled off and was rather decreased on the mite-release leaf at the time of mite emigration. Comparing the changing pattern of the relative degree of aggregation (m ^*/m) measured in two different units between these two species, the pattern ofm ^*/m in 1 cm² on the mite release leaf inO. ununguis resembled that of the unit of leaves on the sapling inP. citri. This result as well as behavioural observations indicate that migration ofO. ununguis is the movement from leaf to leaf and that ofP. citri from sapling to sapling. It is, therefore, concluded that the boundary of the microhabitat is a single leaf forO. ununguis but sapling or foliage forP. citri. This work was presented in Annual Meeting of Jap. Soc. Appl. Ent. Zool., 1981 in Okayama.     

Effective molecular detection of Diaphorina citri Kuwayama (Hemiptera: Liviidae) in bulk insect samples from sticky traps

Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is the principal vector of citrus greening (huanglongbing) disease. Invasion of new areas by the vector increases the risk of further spread of the disease and has economic impacts on the global citrus industry. Effective implementation of vector surveys is essential to contain disease outbreaks. This is especially true in countries such as Japan, where most of the major citrus‐producing areas are free from citrus greening. Recently, vector surveys have been routinely conducted to maintain ‘disease‐free’ and ‘disease‐ and vector‐free’ areas in Japan, and improvement of methods that can detect D. citri in native insect populations is imperative. Here, we developed a method of using conventional and real‐time PCR to detect D. citri among bulk insects captured in sticky traps without the need for preliminary differentiation steps based on morphology. DNA fragments of D. citri were specifically detected by both conventional and real‐time PCR in a mixture of a 10^?3 dilution (ca. 0.008–0.009 ng/μl) of D. citriDNA and 100 ng/μl of bulk insect DNA, indicating that small body parts such as pieces of leg or parts of wings of D. citri were detectable in the bulk insect samples. No misleading amplification of fragments from the other psyllid species and citrus pests we used occurred under our PCR conditions. Our results suggest that the technique is applicable to extensive surveys of D. citri in early warning programmes.     

佛冈县不同类型橘园柑橘木虱的种群动态

[目的] 明确佛冈县不同类型橘园柑橘木虱的种群动态,为当地柑橘木虱及柑橘黄龙病的防治工作提供科学依据。[方法] 通过2015-2016年的系统调查,对广东省佛冈县砂糖橘园的柑橘木虱种群消长动态进行研究,对比分析了不同处理措施对柑橘木虱种群数量的影响。[结果] 1-3月柑橘木虱发生危害较轻,随着嫩梢增多,其种群数量开始上升,6-9月为种群发生高峰期,期间出现多个发生高峰,10月之后种群数量逐渐进入消退期,种群数量维持在较低水平;2015、2016年佛冈县柑橘木虱的季节性消长动态基本一致,但种群发生量存在明显差别;3种不同类型橘园内柑橘木虱种群发生数量也存在显著差异,常年失管橘园柑橘木虱种群发生数量最高,常规水肥管理橘园次之,二者均显著高于常规水肥管理及化学防治橘园。[结论] 化学防治可在一定程度上控制柑橘木虱的种群数量,及时清理失管橘园对控制柑橘木虱和柑橘黄龙病传播至关重要。     

Proteomic maps of subcellular protein fractions of the Asian citrus psyllid Diaphorina citri,the vector of citrus huanglongbing

The Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) is an insect vector that transmits the bacterial pathogen Candidatus Liberibacter asiaticus (CLas) associated with the destructive citrus disease, citrus huanglongbing (HLB). Currently, D. citri is the major target in HLB management, although insecticidal control and disruption of the D. citri–CLas interactions both face numerous challenges. The present study reports the subcellular proteomic profiles of D. citri, encompassing the three main subcellular protein fractions: cytosol, mitochondria and microsomes. After optimization, subcellular proteins of both high and low abundance are obtained by two‐dimensional gel electrophoresis (2‐DE). A total of 1170 spots are detected in the 2‐DE gels of the three subcellular fractions. One hundred and sixty‐four differentially expressed proteins are successfully identified using liquid chromatography‐dual mass spectroscopy. An efficient protocol for subcellular protein fractionation from D. citri is established and a clear protein separation is achieved with the chosen protein fractionation protocol. The identified cytosolic proteins are mainly metabolic enzymes, whereas a large portion of the identified proteins in the mitochondrial and microsomal fractions are involved in ATP biosynthesis and protein metabolism, respectively. Protein–protein interaction networks are predicted for some identified proteins known to be implicated in pathogen–vector interactions, such as actin, tubulin and ATP synthase, as well as insecticide resistance, such as the cytochrome P450 superfamily. The findings should provide useful information to help identify the mechanism responsible for the CLas–D. citri interactions and eventually contribute to D. citri control.     

Guava leaf volatiles and dimethyl disulphide inhibit response of Diaphorina citri Kuwayama to host plant volatiles

The Asian citrus psyllid, Diaphorina citri Kuwayama, vectors the causal pathogen of huanglongbing (HLB), which is likely the most important disease affecting worldwide citrus production. Interplanting citrus with guava, Psidium guajava L., was reported to reduce D. citri populations and incidence of HLB. We describe a series of investigations on the response of D. citri to citrus volatiles with and without guava leaf volatiles and to synthetic dimethyl disulphide (DMDS), in laboratory olfactometers and in the field. Volatiles from guava leaves significantly inhibited attraction of D. citri to normally attractive host‐plant (citrus) volatiles. A similar level of inhibition was recorded when synthetic DMDS was co‐released with volatiles from citrus leaves. In addition, the volatile mixture emanating from a combination of intact citrus and intact guava leaves induced a knock‐down effect on adult D. citri. Compounds similar to DMDS including dipropyl disulphide, ethyl‐1‐propyl disulphide, and diethyl disulphide did not affect the behavioural response of D. citri to attractive citrus host plant volatiles. Head‐space volatile analyses were conducted to compare sulphur volatile profiles of citrus and guava, used in our behavioural assays, with a gas chromatography‐pulsed flame photometric detector. DMDS, produced by wounded guava in our olfactometer assays, was not produced by similarly wounded citrus. The airborne concentration of DMDS that induced the behavioural effect in the 4‐choice olfactometer was 107 pg/ml. In a small plot field experiment, populations of D. citri were significantly reduced by deployment of synthetic DMDS from polyethylene vials compared with untreated control plots. Our results verify that guava leaf volatiles inhibit the response of D. citri to citrus host plant volatiles and suggest that the induced compound, DMDS, may be partially responsible for this effect. Also, we show that field deployment of DMDS reduces densities of D. citri and thus may have potential as a novel control strategy.     

Suitability of native North American Rutaceae to serve as host plants for the Asian citrus psyllid (Hemiptera: Liviidae)

The Asian citrus psyllid, Diaphorina citri Kuwayama, a vector of citrus huanglongbing, is now present in all citrus‐producing states in the USA and Mexico. In addition to citrus, the insect can reproduce on several other plant species in the Rutaceae family; orange jasmine (Murraya spp.) and curry leaf (Bergera koenigii) are among its preferred hosts. There are several indigenous Rutaceae species in North America, and some are popular ornamentals. A study was therefore initiated to determine the suitability of some of these plants for feeding and development of the psyllid in choice and no‐choice experiments. D. citri was found to reproduce successfully on Choisya ternata, C. arizonica and Helietta parvifolia in no‐choice tests, but preferentially selected orange jasmine and curry leaf for feeding and reproduction, in choice tests. On Amyris madrensis, A. texana and Zanthoxylum fagara, adult psyllids laid eggs which hatched, but no successful nymphal development was recorded beyond the first instars. No oviposition was recorded on Esenbeckia berlandieri, Ptelea trifoliata and Casimiroa tetrameria, although adult psyllids were able to survive on these species for several days. Results showed that C. ternata, C. arizonica and H. parvifolia can serve host plants of D. citri and this constitutes the first report of these plants serving as host for D. citri. The findings of the present study suggest that native rutaceous host plants can serve as host plants and thus affect D. citri population dynamics and the epidemiology of Huanglongbing, the deadly citrus greening disease whose pathogen is vectored by D. citri. Thus, area‐wide management of this pest also should target these riparian habitats where these host plants are present with D. citri biological control agents for sustainable management of this pest.     

Best linear unbiased prediction of host-range of the facultative parasite Colletotrichum gloeosporioides f. sp. salsolae, a potential biological control agent of Russian thistle

Russian thistle or tumbleweed (Salsola tragus L.) is an introduced invasive weed in N. America. It is widely distributed in the US and is a target of biological control efforts.The fungus Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. in Penz. f. sp. salsolae (CGS) is a facultative parasite under evaluation for classical biological control of this weed. Host-range tests were conducted with CGS in quarantine to determine whether the fungus is safe to release in N. America. Ninetytwo accessions were analyzed from 19 families: Aizoaceae, Alliaceae, Amaranthaceae, Apiaceae, Asteraceae, Brassicaceae, Cactaceae, Campanulaceae, Chenopodiaceae, Cucurbitaceae, Cupressaceae, Fabaceae, Malvaceae, Nyctaginaceae, Phytolaccaceae, Poaceae, Polygonaceae, Sarcobataceae, and Solanaceae and 10 tribes within the Chenopodiaceae: Atripliceae, Beteae, Camphorosmeae, Chenopodieae, Corispermeae, Halopepideae, Polycnemeae, Salicornieae, Salsoleae, and Suaedeae. These included 62 genera and 120 species. To facilitate interpretation of results, disease reaction data were combined with a relationship matrix derived from internal transcribed spacer DNA sequences and analyzed with mixed model equations to produce Best Linear Unbiased Predictors (BLUPs) for each species. Twenty-nine species (30 accessions) from seven closely-related Chenopodiaceae tribes had significant levels of disease severity as indicated by BLUPs, compared to six species determined to be susceptible with least squares means estimates. The 29 susceptible species were: 1 from Atripliceae, 4 from Camphorosmeae, 1 from Halopepideae, 2 from Polycnemeae, 6 from Salicornieae, 8 from Salsolae, and 7 from Suaedeae. Most species in the genus Salsola, which are all introduced and weedy, were very susceptible and damaged by CGS. Statistical comparisons and contrasts of BLUPs indicated that these Salsola species were significantly more susceptible than non-target species, including 15 species from relatives in the closely-related genera Bassia (=Kochia), Nitrophila, Salicornia, Sarcocornia, and Suaeda. Of the 29 susceptible species, 10 native or commercially important species in N. America were identified as needing additional tests to determine the extent of any damage caused by infection.     

Effects of different temperature regimes on survival of Diaphorina citri and its endosymbiotic bacterial communities

The Asian citrus psyllid, Diaphorina citri, is a major pest of citrus and vector of citrus greening (huanglongbing) in Asian. In our field‐collected psyllid samples, we discovered that Fuzhou (China) and Faisalabad (Pakistan), populations harbored an obligate primary endosymbiont Candidatus Carsonella (gen. nov.) with a single species, Candidatus Carsonella ruddii (sp. nov.) and a secondary endosymbiont, Wolbachia surface proteins (WSP) which are intracellular endosymbionts residing in the bacteriomes. Responses of these symbionts to different temperatures were examined and their host survival assessed. Diagnostic PCR assays showed that the endosymbionts infection rates were not significantly reduced in both D. citri populations after 24 h exposure to cold or heat treatments. Although quantitative PCR assays showed significant reduction of WSP relative densities at 40°C for 24 h, a substantial decrease occurred as the exposure duration increased beyond 3 days. Under the same temperature regimes, Ca. C. ruddii density was initially less affected during the first exposure day, but rapidly reduced at 3–5 days compared to WSP. However, the mortality of the psyllids increased rapidly as exposure time to heat treatment increased. The responses of the two symbionts to unfavorable temperature regimes highlight the complex host‐symbionts interactions between D. citri and its associated endosymbionts.     

Characterization of electrical penetration graphs of the Asian citrus psyllid, Diaphorina citri, in sweet orange seedlings

Detailed information on probing behavior of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is critical for understanding the transmission process of phloem‐limited bacteria (Candidatus Liberibacter spp.) associated with citrus ‘huanglongbing’ by this vector. In this study, we investigated stylet penetration activities of D. citri on seedlings of Citrus sinensis (L.) Osbeck cv. Pêra (Rutaceae) by using the electrical penetration graph (EPG‐DC system) technique. EPG waveforms were described based on amplitude, frequency, voltage level, and electrical origin of the observed traces during stylet penetration into plant tissues. The main waveforms were correlated with histological observations of salivary sheath termini in plant tissues, to determine the putative location of stylet tips. The behavioral activities were also inferred based on waveform similarities in relation to other Sternorrhyncha, particularly aphids and whiteflies. In addition, we correlated the occurrence of specific waveforms with the acquisition of the phloem‐limited bacterium Ca. Liberibacter asiaticus by D. citri. The occurrence of a G‐like xylem sap ingestion waveform in starved and unstarved psyllids was also compared. By analyzing 8‐h EPGs of adult females, five waveforms were described: (C) salivary sheath secretion and other stylet pathway activities; (D) first contact with phloem (distinct from other waveforms reported for Sternorrhyncha); (E1) putative salivation in phloem sieve tubes; (E2) phloem sap ingestion; and (G) probably xylem sap ingestion. Diaphorina citri initiates a probe with stylet pathway through epidermis and parenchyma (C). Interestingly, no potential drops were observed during the stylet pathway phase, as are usually recorded in aphids and other Sternorrhyncha. Once in C, D. citri shows a higher propensity to return to non‐probing than to start a phloem or xylem phase. Several probes are usually observed before the phloem phase; waveform D is observed upon phloem contact, always immediately followed by E1. After E1, D. citri either returns to pathway activity (C) or starts phloem sap ingestion, which was the longest activity observed.     

Selection of Bacillus thuringiensis strains in citrus and their pathogenicity to Diaphorina citri (Hemiptera: Liviidae) nymphs

Bacillus thuringiensis (Bt) toxins are effective in controlling insect pests either through the spraying of products or when expressed in transgenic crops. The discovery of endophytic Bt strains opened new perspectives for studies aimed at the control of sap‐sucking insects, such as the Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae), a vector of “Candidatus Liberibacter spp.,” associated with citrus huanglongbing (HLB). In this study, translocation of endophytic Bt strains in citrus seedlings inoculated with Bt suspension delivered by soil‐drench, and their systemic pathogenicity to D. citri nymphs were investigated. The pathogenicity of three wild‐type Bt strains against D. citri third‐instar nymphs was demonstrated. Among the 10 recombinant strains tested (each of them harboring a single cry or cyt gene), 3 can be highlighted, causing 42%–77% and 66%–90% nymphal mortality at 2 and 5 d after inoculation, respectively. The isolation of Bt cells from young citrus shoots and dead nymphs, and PCR performed with specific primers, confirmed the involvement of the Bt strains in the psyllid mortality. This is the first report showing the translocation of Bt strains from citrus seedling roots to shoots and their potential to control D. citri nymphs that fed on these soil‐drench inoculated seedlings. The Bt strains that caused the highest mortality rates have the potential to be used as bioinsecticides to control D. citri and the identified genes can be used for the production of transgenic Bt citrus.