Injection–Infiltration of Attached Grapefruit with Xanthomonas citri subsp. citri to Evaluate Seasonal Population Dynamics in Citrus Canker Lesions

To study population dynamics of Xanthomonas citri subsp. citri (Xcc) in citrus canker lesions on fruit, a needle‐free injector was used for infiltration of bacterial inoculum into fruit in situ on mature ‘Ruby Red’ grapefruit (Citrus paradisi Macf.) trees in Florida. Inoculations of Xcc at 10⁵ colony‐forming units (cfu) per ml were conducted in 2012 and 2013 on attached fruit varying from 15 mm to 90 mm in diameter. Inoculations were repeated every 2–3 weeks until the fruit were no longer injectable. On fruit less than 40 mm in size, erumpent lesions formed within 2 weeks of inoculation and expanded 1–9 mm in diameter from 30 to 120 days postinoculation (dpi). Xcc populations in lesions were 6–8 log cfu per lesion at 30 dpi and maintained this population up to 90 dpi. By 120 dpi, Xcc populations declined 1–3 log units as rainfall and temperature decreased in September–October. Xcc populations declined to ~3 log cfu per lesion after 120 dpi in November 2012 and 2013, whereas the population resurged to 5 log cfu per lesion after 180 dpi in January–February 2014.     

Engineering canker‐resistant plants through CRISPR/Cas9‐targeted editing of the susceptibility gene CsLOB1 promoter in citrus

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is severely damaging to the global citrus industry. Targeted editing of host disease‐susceptibility genes represents an interesting and potentially durable alternative in plant breeding for resistance. Here, we report improvement of citrus canker resistance through CRISPR/Cas9‐targeted modification of the susceptibility gene CsLOB1 promoter in citrus. Wanjincheng orange (Citrus sinensis Osbeck) harbours at least three copies of the CsLOB1^G allele and one copy of the CsLOB1^? allele. The promoter of both alleles contains the effector binding element (EBE_PthA4), which is recognized by the main effector PthA4 of Xcc to activate CsLOB1 expression to promote citrus canker development. Five pCas9/CsLOB1sgRNA constructs were designed to modify the EBE_PthA4 of the CsLOB1 promoter in Wanjincheng orange. Among these constructs, mutation rates were 11.5%–64.7%. Homozygous mutants were generated directly from citrus explants. Sixteen lines that harboured EBE_PthA4 modifications were identified from 38 mutant plants. Four mutation lines (S2‐5, S2‐6, S2‐12 and S5‐13), in which promoter editing disrupted CsLOB1 induction in response to Xcc infection, showed enhanced resistance to citrus canker compared with the wild type. No canker symptoms were observed in the S2‐6 and S5‐13 lines. Promoter editing of CsLOB1^G alone was sufficient to enhance citrus canker resistance in Wanjincheng orange. Deletion of the entire EBE_PthA4 sequence from both CsLOB1 alleles conferred a high degree of resistance to citrus canker. The results demonstrate that CRISPR/Cas9‐mediated promoter editing of CsLOB1 is an efficient strategy for generation of canker‐resistant citrus cultivars.     

Recent advances in the understanding of Xanthomonas citri ssp. citri pathogenesis and citrus canker disease management

Taxonomic status : Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Xanthomonadales; Family Xanthomonadaceae; Genus Xanthomonas; Species Xanthomonas citri ssp. citri (Xcc). Host range : Compatible hosts vary in their susceptibility to citrus canker (CC), with grapefruit, lime and lemon being the most susceptible, sweet orange being moderately susceptible, and kumquat and calamondin being amongst the least susceptible. Microbiological properties : Xcc is a rod‐shaped (1.5–2.0 × 0.5–0.75 µm), Gram‐negative, aerobic bacterium with a single polar flagellum. The bacterium forms yellow colonies on culture media as a result of the production of xanthomonadin. Distribution : Present in South America, the British Virgin Islands, Africa, the Middle East, India, Asia and the South Pacific islands. Localized incidence in the USA, Argentina, Brazil, Bolivia, Uruguay, Senegal, Mali, Burkina Faso, Tanzania, Iran, Saudi Arabia, Yemen and Bangladesh. Widespread throughout Paraguay, Comoros, China, Japan, Malaysia and Vietnam. Eradicated from South Africa, Australia and New Zealand. Absent from Europe.     

Efficacy of different copper compounds in the control of Xanthomonas citri subsp. citri pathotypes A and A*

Citrus canker disease is one of the most devastating diseases that attacks citrus, especially limes in the Southern parts of Iran, and is caused by Xanthomonas citri subsp. citri (Xcc). The efficacy of several formulations of copper compounds including Bordeaux mixture, copper oxychloride and copper sulphate in controlling Xcc in Key lime was estimated in vitro and in planta using artificial inoculation. Specific primers were used to detect copper-resistant genes copA, copB and copL in 30 isolates of Xcc. The copA and copL genes were present in all isolates, and copB was detected only in 6 strains. In this study, we observed a very good in vitro growth inhibition activity of copper compounds against Xcc pathotype A. S14 strain (pathotype A^*) was the sole isolate that grew on media amended with 2/4 mM of Bordeaux mixture, copper oxychloride and copper sulphate. All other strains (pathotype A) failed to grow on media amended with this concentration. Bordeaux mixture exhibited high efficacy in controlling Xcc in both conditions. However, there were no significant differences in the efficacy of copper oxychloride and copper sulphate at 1.2 mM concentration in planta. A significantly minimum canker necrotic spot and highest disease control was achieved with Bordeaux mixture and copper oxychloride. There was a significant difference in disease severity of the type strain LMG9322 (pathotype A) and Xcc strain S14 (pathotype A^*). Our experiments showed that Bordeaux mixture exhibited satisfactory efficacy in controlling the causal agent of citrus canker.     

The role as inoculum sources of Xanthomonas citri pv. citri surviving on the infected Satsuma mandarin fruits

Importing citrus fruits infected by Asiatic citrus canker caused by Xanthomonas citri pv. citri (Xcc) can act as an inoculum source for the disease epidemic in citrus canker-free countries. In this study, the pathogenicity of the causal agent of Asiatic citrus canker surviving on infected Satsuma mandarin fruits was evaluated. The washing solution of infected Satsuma mandarin fruits did not cause lesion formation on the citrus leaves. However, a typical citrus canker lesion was formed on the leaves after inoculation with higher concentrations of the inoculum from the washing solution (washing solution II). It indicated that the pathogenicity of the citrus canker surviving on the symptomatic Satsuma mandarin fruits was not changed. Scanning electron microscopic observation showed that the numbers of bacterial cells on the leaves of Satsuma mandarin which inoculated with the washing solution directly (washing solution I) was less compared to those of leaves inoculated with the washing solution II. This result spports that the pathogenicity of Xcc surviving on Satsuma mandarin fruits may not be changed but that the sucessful infection of citrus caker may depend on the concentration of the inoculum.     

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.     

Biocontrol of citrus bacterial canker caused by Xanthomonas citri subsp. citri by Bacillus velezensis

Microorganisms with biocontrol capabilities against plant pathogens are considered as one of the most promising approaches for healthy crop management. In this study, ethyl acetate extracts of 25 Bacillus strains were investigated for their antagonistic effect on Xanthomonas citri subsp. citri (Xcc), which causes the citrus bacterial canker (CBC) disease. Among them, 21 strains exerted antibacterial activity against wild-type Xcc strains. Based on the strength of the antibacterial activity, nine Bacillus strains were selected for 16S rRNA analysis. 16S rRNA sequence homology revealed that several strains were closely related to B. velezensis, where strains with no antibacterial activity grouped as the soil-associated community of B. amyloliquefaciens. B. velezensis Bv-21 exhibited the highest antibacterial activity against wild type and streptomycin resistant Xcc with inhibition zones of 22.91 ± 0.45 and 20.28 ± 0.53, respectively. Furthermore, B. velezensis Bv-21 strain was tested for biocontrol activity against a streptomycin-resistant XccM4 in detached susceptible citrus leaves. The strain reduced the incidence of CBC by 26.30% and pathogen density of XccM4 by 81.68% over control. The results of the study strongly suggest that B. velezensis can be used as an effective and eco-friendly biocontrol agent either by itself or as an active compound, against both, the wild-type and streptomycin-resistant Xcc.     

Cryptolaemus montrouzieri as a predator of the striped mealybug,Ferrisia virgata,reared on two hosts

The striped mealybug, Ferrisia virgata (Cockerell) (Hemiptera: Pseudococcidae), is a cosmopolitan pest of a variety of agricultural crops including cotton. To investigate the biological control potential of the predatory ladybird Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae) against this pest, we evaluated its developmental and reproductive fitness when feeding on F. virgata reared on pumpkin fruits or on cotton leaves and compared this to a diet of Planococcus citri Risso (Hemiptera: Pseudococcidae) reared on pumpkin fruits. F. virgata and P. citri reared on pumpkins were equally suitable prey for the pre‐imaginal stages of C. montrouzieri. Duration of total immature development was 1 day longer in C. montrouzieri offered F. virgata reared on cotton as compared with F. virgata or P. citri reared on pumpkin, whereas no significant difference was observed in survival rates. Diet significantly influenced the reproductive fitness of C. montrouzieri. Females offered P. citri reared on pumpkin had significantly shorter pre‐oviposition periods and higher fecundity and fertility than those given F. virgata reared on pumpkin or cotton leaves. F. virgata grown on cotton leaves supported the reproduction of C. montrouzieri better than F. virgata reared on pumpkin. Our study established that C. montrouzieri can successfully complete its development and reproduction when fed exclusively on F. virgata and indicates its potential as a biological control agent of this emerging cotton pest.     

Ingestible insecticides for spotted wing Drosophila control: a polyol,Erythritol, and an insect growth regulator,Lufenuron

Bioassays tested insecticidal activity of Erythritol from the nutritive sweetener, Truvia, and an insect growth regulator, Lufenuron, against life stages (eggs, larvae, pupae, adults) of Drosophila melanogaster (Meigen) and Drosophila suzukii (Matsumura), the spotted wing Drosophila (SWD). These compounds were chosen for their demonstrated acute toxicity to adult and larval Drosophila and potential use on organic fruit farms. D. melanogaster fed on standard Drosophila diet media moistened with water containing known concentrations of Erythritol. Likewise, SWD consumed standard diet media as well as thawed host fruit (blackberries and blueberries) treated with solutions of Erythritol, Lufenuron or both. During the first bioassay, Erythritol at lower concentrations between 0 and 500 mm (~61 000 ppm) in water and mixed with instant diet media increased adult survival from ~80% to 97% for D. melanogaster and SWD. However, from aqueous concentrations ranging from 1750 (~414 000 ppm) to 2000 mm (~244 000 ppm), Erythritol killed 100% of adult Drosophila in culture vials. One hundred per cent mortality for SWD and D. melanogaster occurred at ≥0.5 m (~61 000 ppm) Erythritol added to diet media or topically applied to host fruit. In a second bioassay, 0.013–1.000 ppm of aqueous Lufenuron, a chitin synthase inhibitor, when added to dry diet media prevented 90–99% of SWD from reaching the pupal stage. In another assay, ~67% of SWD eggs or neonates (early first instars) died inside blackberries pre‐treated with (dipped in) a soapy solution of 10 ppm Lufenuron. Pre‐treating blackberry fruit with an Erythritol–Lufenuron mixture reduced SWD brood survival by 99%. Likewise, during our last fruit‐based bioassay, 98% of eggs and neonates died inside blueberries similarly pre‐treated. During the last experiment, Lufenuron in diet media also rendered adult females sterile. Sterility, however, dissipated over 7 days once females began feeding on a Lufenuron‐free diet media.     

Evaluation of the antibiotic activity of extracellular compounds produced by the Pseudomonas strain against the Xanthomonas citri pv. citri 306 strain

In this work, we evaluated the antibiotic activity of metabolites produced by the Pseudomonas sp. LV strain and their effects on the cell morphology of the Xanthomonas citri pv. citri 306 strain (Xcc 306), which causes citrus canker lesions. The LV strain was cultivated, centrifuged, a cell-free supernatant was treated with dichloromethane and then concentrated, frozen in liquid nitrogen and lyophilized. The dichloromethane phase (DP) was fractionated by vacuum liquid chromatography (VLC) using six organic solvents with a crescent polarity. The antibiotic activity of the DP and all the fractions from VLC were tested against Xcc 306 and only the F3 fraction showed antimicrobial activity. The antibiotic activity of F3 was determined by minimum inhibitory concentration and the action on the cell morphology of Xcc 306 carried out in glass tubes with cell suspensions plus F3 solution sampled at three different times (one, three and six hours). The effects were analyzed by electron microscopy. Both the DP and F3 showed antibiotic activity against Xcc 306 in in vitro experiments. Electron microscopy showed that the F3 fraction completely disrupted the cell integrity after six hours. In a greenhouse experiment, the DP and F3 fraction (highly effective in in vitro experiments), reduced the formation of lesions by approximately 80% and 94%, respectively.     

HrpM is involved in glucan biosynthesis,biofilm formation and pathogenicity in Xanthomonas citri ssp. citri

Xanthomonas citri ssp. citri (Xcc) is the causal agent of citrus canker. This bacterium develops a characteristic biofilm on both biotic and abiotic surfaces. A biofilm‐deficient mutant was identified in a screening of a transposon mutagenesis library of the Xcc 306 strain constructed using the commercial Tn5 transposon EZ‐Tn5 <KAN‐2> Tnp Transposome (Epicentre). Sequence analysis of a mutant obtained in the screening revealed that a single copy of the EZ‐Tn5 was inserted at position 446 of hrpM, a gene encoding a putative enzyme involved in glucan synthesis. We demonstrate for the first time that the product encoded by the hrpM gene is involved in β‐1,2‐glucan synthesis in Xcc. A mutation in hrpM resulted in no disease symptoms after 4 weeks of inoculation in lemon and grapefruit plants. The mutant also showed reduced ability to swim in soft agar and decreased resistance to H ₂ O ₂ in comparison with the wild‐type strain. All defective phenotypes were restored to wild‐type levels by complementation with the plasmid pBBR1‐MCS containing an intact copy of the hrpM gene and its promoter. These results indicate that the hrpM gene contributes to Xcc growth and adaptation in its host plant.     

Oviposition preferences in Grapholita molesta: the relative importance of visual and olfactory cues

The oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae), is a major pest of fruit trees worldwide. Females oviposit on the young leaves of peach [Prunus persica (L.) Batsch (Rosaceae)] shoots at twilight, when light intensity changes markedly. Previous researches have shown that the oviposition response of this moth is guided particularly by visual and olfactory cues from host plants. However, the relative importance of visual and olfactory cues in their oviposition preference is largely unknown. As a crepuscular moth, how do choices change as light intensity drops rapidly from day to night? In the present study, through two‐choice behavioural experiments, the oviposition responses of G. molesta to visual and olfactory cues (alone or in combination) from peach shoots were tested at four light intensities ranging from daylight to starlight. Grapholita molesta showed similar oviposition responses to plant cues under 1 000, 100, 1 and 0.01 mW m^?2 illumination. Olfactory cues from peach shoots attracted significantly more oviposition than the negative control, whereas visual cues alone did not. Furthermore, olfactory cues were more attractive than visual cues, and no significant interaction was observed between the responses to the two cues. Our findings indicate that G. molesta females mainly rely on olfactory cues to recognize oviposition sites, regardless of differences in light intensity. These results do not provide evidence for attraction to visual cues, but the possibility that the brightness of leaves might be used to guide oviposition is discussed.     

Translocation and multiplication ofSpiroplasma citri in turnip (Brassica rapa)

Following inoculation of designated leaves of turnip plants withSpiroplasma citri byCirculifer tenellus, spiroplasmas were cultured first from roots (four days) and then from youngest leaves (eight days), but almost never from oldest leaves. In experiments using enzyme-linked immunosorbent assay to monitor changes in titer in turnip leaves during the course of plant infection,S. citri was detected seven days after inoculation and reached peak titers of 10¹⁰–10¹¹ colony-forming units/g 12–20 days after inoculation, declining thereafter. Spiroplasmas were detected 5–9 days before symptoms appeared.     

Leaf age affects the efficacy of insecticides to control Asian citrus psyllid,Diaphorina citri (Hemiptera: Liviidae)

Huanglongbing (HLB), the most destructive citrus disease worldwide, is associated with the bacteria transmitted by the psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae). One of the main tactics used to manage this disease is the chemical control of D. citri. In this study, the efficacy of four insecticides commonly used by the citrus growers against the D. citri adults was assessed in two vegetative growth stages of flush: young shoots (~ 10 cm in length) and mature leaves. The bioassays were carried out under the greenhouse and field conditions. The insecticides bifenthrin, dimethoate, imidacloprid and thiamethoxam were tested at the recommended rates of 15, 450, 40 and 25 mg/L of water, respectively. The insecticides were sprayed onto the leaves of each stage until runoff. Psyllid mortality was assessed at different periods after insecticide application (0, 1 and 2 weeks). In both greenhouse and field trees, the period of control was shorter in the young shoots than in the mature leaves. In addition, the efficacy of insecticides rapidly decreased in the young shoots over time, mainly for imidacloprid and bifenthrin (less than 1 week). Thus, these results suggest that the number of spray applications to control D. citri should be more frequent during the flushing period.     

Lactose consuming strains of Xanthomonas citri subsp. citri (Xcc) insight into the emergence of natural field resources for xanthan gum production

Xanthomonas genus possesses a low level of β-galactosidase gene expression and is therefore unable to produce xanthan gum in lactose-based media. In this study, we report the emergence of some natural field strains of Xanthomonas citri subsp. citri (Xcc) capable to use lactose as a sole carbon source to produce xanthan gum. From 210 Xcc strains isolated from key lime (C. aurantifolia), 27 showed the capacity to grow on lactose containing medium. Xcc lactose consuming strains demonstrated a good level of xanthan production. Amongst all, NIGEBK37 produced the greatest (14.62 g/l) amount of xanthan gum in experimental laboratory conditions. By evaluating the viscosity of the biopolymer at 25 °C, it was demonstrated that xanthan synthesized by strain NIGEBK37 has the highest viscosity (44,170.66 cP). Our results were indicative for the weakness of a commercial strain of Xanthomonas campestris pv. Campestris DSM1706 (Xcc/DSM1706) to produce xanthan in lactose containing medium.     

Sex‐specific responses of Asian citrus psyllid to volatiles of conspecific and host‐plant origin

Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is the primary vector of Candidatus Liberibacter spp. bacteria that cause citrus greening, a disease of worldwide importance. Olfactometry was employed to test responses of D. citri to odours from intact citrus plants (Mexican lime, Citrus aurantifolia, sour orange, Citrus aurantium, Marsh grapefruit, Citrus paradisi and Valencia orange, Citrus sinensis), citrus plants previously infested with D. citri, and odours of conspecifics including nymphs, adult insects of same and opposite sex, and their products (honeydew), both alone and in combination. In contrast to other studies, psyllids of both sexes were attracted to volatiles of undamaged Mexican lime leaves, whereas undamaged grapefruit attracted only females, and leaves of Valencia and sour orange did not attract either sex. All four plant species attracted female psyllids when previously infested, but only Mexican lime and sour orange‐attracted males. Thus, Citrus species appear to vary in the production of both constituitive and induced volatiles that attract adult psyllids. Volatiles emitted by nymphs did not attract either sex, but psyllid honeydew was attractive to males, likely due to female pheromone residues. Males oriented to the odour of females, whereas the reverse was not true, and neither males nor females oriented to same‐sex volatiles. The addition of conspecific cues (adults, nymphs or honeydew) did not increase female attraction to previously infested leaves, but male response was increased by the presence of adults and honeydew, regardless of plant species. Thus, female psyllids appear to orient more strongly to volatiles of plant origin, whereas males respond more strongly to cues emanating from females and conspecific excretions. These results suggest that female psyllids drive the initial colonization of host plants, whereas males orient to females and infested plants. Identification of the specific volatiles involved may permit their use in monitoring and management of this pest.     

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.