Healthy Citrus Research Articles

Development of a Tandem Repeat-Based Polymerase Chain Displacement Reaction Method for Highly Sensitive Detection of 'Candidatus Liberibacter asiaticus'.

Huanglongbing (HLB) is one of the most destructive diseases in citrus production worldwide. Early detection of HLB pathogens can facilitate timely removal of infected citrus trees in the field. However, low titer and uneven distribution of HLB pathogens in host plants make reliable detection challenging. Therefore, the development of effective detection methods with high sensitivity is imperative. This study reports the development of a novel method, tandem repeat-based polymerase chain displacement reaction (TR-PCDR), for the detection of 'Candidatus Liberibacter asiaticus', a widely distributed HLB-associated bacterium. A uniquely designed primer set (TR2-PCDR-F/TR2-PCDR-1R) and a thermostable Taq DNA polymerase mutant with strand displacement activity were used for TR-PCDR amplification. Performed in a regular thermal cycler, TR-PCDR could produce more than two amplicons after each amplification cycle. Sensitivity of the developed TR-PCDR was 10 copies of target DNA fragment. The sensitive level was proven to be 100× higher than conventional PCR and similar to real-time PCR. Data from the detection of 'Ca. L. asiaticus' with filed samples using the above three methods also showed similar results. No false-positive TR-PCDR amplification was observed from healthy citrus samples and water controls. These results thereby illustrated that the developed TR-PCDR method can be applied to the reliable, highly sensitive, and cost-effective detection of 'Ca. L. asiaticus'.

Characterization of Antimicrobial-Producing Beneficial Bacteria Isolated from Huanglongbing Escape Citrus Trees.

The microbiome associated with crop plants has a strong impact on their health and productivity. Candidatus Liberibacter asiaticus (Las), the bacterial pathogen responsible for Huanglongbing (HLB) disease, lives inside the phloem of citrus plants including the root system. It has been suggested that Las negatively affects citrus microbiome. On the other hand, members of citrus microbiome also influence the interaction between Las and citrus. Here, we report the isolation and characterization of multiple putative beneficial bacteria from healthy citrus rhizosphere. Firstly, six bacterial strains showing antibacterial activity against two bacteria closely related to Las: Agrobacterium tumefaciens and Sinorhizobium meliloti were selected. Among them, Burkholderia metallica strain A53 and Burkholderia territorii strain A63 are within the β-proteobacteria class, whereas Pseudomonas granadensis strain 100 and Pseudomonas geniculata strain 95 are within the γ-proteobacteria class. Additionally, two gram-positive bacteria Rhodococcus jialingiae strain 108 and Bacillus pumilus strain 104 were also identified. Secondly, antimicrobial activity against three fungal pathogens: Alternaria alternata, Colletotrichum acutatum, Phyllosticta citricarpa, and two oomycetes: Phytophthora nicotianae and Phytophthora palmivora. Four bacterial strains Burkholderia territorii A63, Burkholderia metallica A53, Pseudomonas geniculata 95, and Bacillus pumilus 104 were shown to have antagonistic activity against the citrus root pathogen Phytophthora nicotianae based on dual culture antagonist assays and compartmentalized petri dish assays. The four selected bacteria were sequenced. Genes involved in phosphate solubilization, siderophore production and iron acquisition, volatile organic compound production, osmoprotection and osmotic tolerance, phytohormone production, antagonism, and nutrient competition were predicted and discussed related to the beneficial traits.

Knowledge, Attitude and Behaviour of Farmer on Integrated Crop Management for Healthy Citrus Orchard (ICMHCO) in Gianyar District

One cause of low productivity and quality of citrus in Indonesia is not free of CVPD (Citrus Vein Phloem Degeneration) or Huang Lung Bin (HLB) disease in several citrus production centers. Integrated Crop Management for Healthy Citrus Orchard (ICMHCO) is a HLB control strategy consisted of (a) the used of labeled and free diseases planting materials, (b) pest and diseases control especially for the HLB vector, (c) good field sanitation, (d) optimum cultural practices, and (e) field management consolidation. The research objective was to determine the knowledge, attitudes and behavior of farmers in the control HLB. The research was conducted in the village of Pupuan, Tegallalang Sub-district, Gianyar District on August 2015, using a survey of 30 farmers of respondents. Data were analyzed descriptively uses the cross tabulation. The results showed that the knowledge of farmers on ICMHCO could be classified as moderate, relatively neutral attitude of farmers and farmers' behavior is low. Efforts should be made to improve the implementation of ICMHCO, among others, by increasing the availability of seed labeled and free of disease and other production inputs, training and institutional capacity building of farmer groups. Keywords: knowledge; attitude; behavior; citrus farmer

Protected Fresh Grapefruit Cultivation Systems: Antipsyllid Screen Effects on Plant Growth and Leaf Transpiration, Vapor Pressure Deficit, and Nutrition

Completely enclosed screen houses can physically exclude contact between the asian citrus psyllid [ACP (Diaphorina citri)] and young, healthy citrus (Citrus sp.) trees and prevent huanglongbing (HLB) disease development. The current study investigated the use of antipsyllid screen houses on plant growth and physiological parameters of young ‘Ray Ruby’ grapefruit (Citrus ×paradisi) trees. We tested two coverings [enclosed screen house and open-air (control)] and two planting systems (in-ground and container-grown), with four replications arranged in a split-plot experimental design. Trees grown inside screen houses developed larger canopy surface area, canopy surface area water use efficiency (CWUE), leaf area index (LAI) and LAI water use efficiency (LAIWUE) relative to trees grown in open-air plots (P < 0.01). Leaf water transpiration increased and leaf vapor pressure deficit (VPD) decreased in trees grown inside screen houses compared with trees grown in the open-air plots. CWUE was negatively related to leaf VPD (P < 0.01). Monthly leaf nitrogen concentration was consistently greater in container-grown trees in the open-air compared with trees grown in-ground and inside the screen houses. However, trees grown in-ground and inside the screen houses did not experience any severe leaf N deficiencies and were the largest trees, presenting the highest canopy surface area and LAI at the end of the study. The screen houses described here provided a better growing environment for in-ground grapefruit because the protective structures accelerated young tree growth compared with open-air plantings while protecting trees from HLB infection.

Huanglongbing impairs the rhizosphere-to-rhizoplane enrichment process of the citrus root-associated microbiome

BackgroundRoots are the primary site for plant-microbe interactions. Among the three root-associated layers (i.e., rhizosphere, rhizoplane, and endorhiza), the rhizoplane is a key component serving a critical gating role that controls microbial entry into plant roots. The microbial communities colonizing the three layers are believed to be gradually enriched from the bulk soil inoculum. However, it is unknown how this enrichment process, particularly the rhizosphere to rhizoplane step, is affected by biotic stresses, such as disease. In this study, we address this question using the citrus root-associated microbiome as a model.ResultsWe identified the rhizosphere-to-rhizoplane-enriched taxonomic and functional properties of the citrus root-associated microbiome and determined how they were affected by Huanglongbing (HLB), a severe systemic disease caused by Candidatus Liberibacter asiaticus, using metagenomic and metatranscriptomic approaches. Multiple rhizoplane-enriched genera were identified, with Bradyrhizobium and Burkholderia being the most dominant. Plant-derived carbon sources are an important driving force for the enrichment process. The enrichment of functional attributes, such as motility, chemotaxis, secretion systems, and lipopolysaccharide (LPS) synthesis, demonstrated more active microbe-plant interactions on the rhizoplane than the rhizosphere. We observed that HLB impaired the rhizosphere-to-rhizoplane enrichment process of the citrus root-associated microbiome in three ways: (1) by decreasing the relative abundance of most rhizoplane-enriched genera; (2) by reducing the relative abundance and/or expression activity of the functional attributes involved in microbe-plant interactions; and (3) by recruiting more functional features involved in autotrophic life cycle adaptation, such as carbon fixation and nitrogen nitrification in the HLB rhizoplane microbiome. Finally, our data showed that inoculation of Burkholderia strains isolated from the healthy citrus root-associated microbiome could trigger the expression of genes involved in induced systemic resistance in inoculated plants.ConclusionsHLB causes decreased relative abundance and/or expression activity of rhizoplane-enriched taxonomic and functional properties, collectively resulting in impaired plant host-microbiome interactions. Manipulation of the citrus root-associated microbiome, for instance, by inoculating citrus roots with beneficial Burkholderia strains, has potential to promote plant health. Our results provide novel insights for understanding the contributions of the community enrichment process of the root-associated microbiome to the plant hosts.

Detection of Green Mould Rot Infection of Citrus Fruit by X-ray Scanning Non-Destructive Technology

<p><strong>ABSTRACT</strong> <strong></strong></p><p>Green mould rot is important post-harvest fungal disease of citrus. It is caused by <em>Penicillium digitatum. </em>To study the detection of fungal infection in citrus fruit by X-ray scanning technique<strong>, </strong>the spore suspension of <em>Penicillium </em><em>digitatum </em>was inoculated by sterilized disposable syringe in citrus fruit in aseptic condition. Healthy citrus without inoculation was treated as control. Infection of green mould rot was detected by X-ray scanning nondestructive technology.</p><p><strong>Key words:</strong> <em>Penicillium digitatum</em>, Citrus, X-ray scanning, Post-harvest disease. </p>

Viable microbial loads on citrus carpoplane during packhouse processing and survival of foodborne pathogens in reconstituted postharvest fungicides

AbstractThis study investigated microbial population shifts on citrus carpoplane and the impact of irrigation and packhouse processing water in four commercial farms. Samples included oranges (n = 450) and water (n = 230). Mean microbial concentrations ranging from 1.6 to 3.6 log colony forming units (CFU)/cm2 were detected on unwashed fruit and were reduced at least by 2.0 log CFU/cm2 for bacteria, 0.8 log CFU/cm2 for fungi and 1.3 log CFU/cm2 for yeasts during washing with disinfectants and/or fungicides. Occasional spikes in concentration (mostly for bacteria) were observed following some packing steps including washing, fungicide treatment, waxing, and final packing. Evaluation of Escherichia coli O157:H7 and S. Typhimurium survival in fungicides typically used during packhouse processing showed them to be susceptible to guazatine, imazalil, philabuster, and ortho‐phenyl phenate. Both tested pathogens were able to survive in thiabendazole, fludioxinil and pyremethanil, except for S. Typhimurium in fludioxinil where it died within 4 hr of incubation.Practical applicationsUnderstanding the impact of current intervention strategies on the ecological balance of the citrus carpoplane may provide a more durable approach to reduced losses and spoilage and in developing crop‐specific management systems for food safety assurance. Our study showed process‐by‐process changes in microbial loads naturally present on healthy citrus fruit at harvest. Washing baths containing a sanitizing agent consistently reduced initial microbial loads on the citrus carpoplane. Further, potential survival of E. coli O157:H7 and S. Typhimurium in currently registered commercial postharvest fungicides, was demonstrated. The pesticide fludioxinil was observed to be effective against, S. Typhimurium providing some potential use as a kill step agent, which, however, needs further investigation.

Latency and Persistence of 'Candidatus Liberibacter asiaticus' in Its Psyllid Vector, Diaphorina citri (Hemiptera: Liviidae).

Although 'Candidatus Liberibacter asiaticus' (Las) is a major pathogen associated with citrus huanglongbing (HLB), some characteristics of transmission by the psyllid vector Diaphorina citri are not fully understood. We examined the latent period and persistence of transmission of Las by D. citri in a series of experiments at 25°C, in which third-instar psyllid nymphs and 1-week-old adults were confined on infected citrus for an acquisition access period (AAP), and submitted to sequential inoculation access periods (IAPs) on healthy citrus seedlings. The median latent period (LP50, i.e., acquisition time after which 50% of the individuals can inoculate) of 16.8 and 17.8 days for psyllids that acquired Las as nymphs and adults, respectively, was determined by transferring single individuals in 48-h IAPs. Inoculation events were intermittent and randomly distributed over the IAPs, but were more frequent after acquisition by nymphs. A minimum latent period of 7 to 10 days was observed by transferring groups of 10 psyllids in 48-h IAPs, after a 96-h AAP by nymphs. Psyllids transmitted for up to 5 weeks, when submitted to sequential 1-week IAPs after a 14-day AAP as nymphs. The long latent period and persistence of transmission are indirect evidences of circulative propagation of Las in D. citri.

A Muscodor strain isolated from Citrus sinensis and its production of volatile organic compounds inhibiting Phyllosticta citricarpa growth

The citrus industry is among the most important worldwide, but citrus plants are affected by a large number of diseases, such as Citrus Black Spot (CBS), caused by the fungus Phyllosticta citricarpa. To identify alternative methods for CBS control, endophytic fungi were isolated by our group from healthy citrus plants in Brazil. Over 400 fungal isolates were obtained, and isolate LGMF1254 was selected based on its inhibitory effect on the growth of P. citricarpa because of the production of volatile organic compounds (VOCs). This isolate was identified as Muscodor sp. by morphological examination and phylogenetic analysis using the internal transcribed spacer (ITS) region: High similarity with M. sutura, M. vitigenus, and M. equiseti was observed. We also sequenced the RPB2 gene, and isolate LGMF1254 showed 99% similarity with M. sutura. To identify this isolate at the species level, the main VOCs were determined, and according to this analysis, LGMF1254 may be classified as an M. sutura strain; however, sequences from regions other than ITS and RPB2 are necessary for conclusive genotyping. The VOCs produced by strain LGMF1254 can be considered an alternative way to control P. citricarpa during the transport of fruits and to prevent the development of CBS signs.

Proteomics analysis reveals novel host molecular mechanisms associated with thermotherapy of 'Ca. Liberibacter asiaticus'-infected citrus plants.

BackgroundCitrus Huanglongbing (HLB), which is linked to the bacterial pathogen ‘Ca. Liberibacter asiaticus’ (Las), is the most devastating disease of citrus plants, and longer-term control measures via breeding or genetic engineering have been unwieldy because all cultivated citrus species are susceptible to the disease. However, the degree of susceptibility varies among citrus species, which has prompted efforts to identify potential Las resistance/tolerance-related genes in citrus plants for application in breeding or genetic engineering programs. Plant exposure to one form of stress has been shown to serendipitously induce innate resistance to other forms of stress and a recent study showed that continuous heat treatment (40 to 42 °C) reduced Las titer and HLB-associated symptoms in citrus seedlings. The goal of the present study was to apply comparative proteomics analysis via 2-DE and mass spectrometry to elucidate the molecular processes associated with heat-induced mitigation of HLB in citrus plants. Healthy or Las-infected citrus grapefruit plants were exposed to room temperature or to continuous heat treatment of 40 °C for 6 days.ResultsAn exhaustive total protein extraction process facilitated the identification of 107 differentially-expressed proteins in response to Las and/or heat treatment, which included a strong up-regulation of chaperones including small (23.6, 18.5 and 17.9 kDa) heat shock proteins, a HSP70-like protein and a ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO)-binding 60 kDa chaperonin, particularly in response to heat treatment. Other proteins that were generally down-regulated due to Las infection but up-regulated in response to heat treatment include RuBisCO activase, chlorophyll a/b binding protein, glucosidase II beta subunit-like protein, a putative lipoxygenase protein, a ferritin-like protein, and a glutathione S-transferase.ConclusionsThe differentially-expressed proteins identified in this study highlights a premier characterization of the molecular mechanisms potentially involved in the reversal of Las-induced pathogenicity processes in citrus plants and are hence proposed targets for application towards the development of cisgenic Las-resistant/tolerant citrus plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0942-x) contains supplementary material, which is available to authorized users.

Feeding behavior of Diaphorina citri and its transmission of ‘Candidatus Liberibacter asiaticus’ to citrus

AbstractHuanglongbing (HLB), also known as citrus greening, is currently the most destructive disease of citrus, responsible for huge economic losses in the world's major citrus production areas. The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), transmits ‘Candidatus Liberibacter asiaticus’ (Clas), the pathogen responsible to cause HLB. Understanding of vector, pathogen, and host plant interactions is important for the management of this vector‐disease complex. We used the direct‐current electrical penetration graph (DC‐EPG) system to evaluate feeding behavior of Clas‐infected D. citri adults, and their potential to transmit the pathogen to healthy citrus, Citrus reticulata Blanco cv. Sunki (Rutaceae), following a 24‐h inoculation access period. Plants were tested for the presence of Clas by qPCR 6 months after inoculation. Findings suggest that inoculation was associated with salivation into the phloem sieve elements (waveform E1). The minimum feeding time for successful transmission by a single adult was 88.8 min, with a minimum E1 duration of 5.1 min. Regression analysis indicated a significant relationship between E1 duration and transmission efficiency. The adults successful in transmitting Clas to healthy citrus were able to penetrate and feed in the phloem much earlier than those which did not transmit. The minimum duration of E1 for a female was shorter than that of a male, but transmission was higher. However, durations of other EPG parameters were not significantly different between male and female. Feeding by single Clas‐infected D. citri adults on 6‐month‐old plants (Sunki) resulted in 23% HLB‐positive plants 6 months after inoculation. Multiple nymphs or adults could transmit the pathogen more efficiently than individual adults in the field, and further enhance the severity of the disease. Effective tactics are warranted to control D. citri and disrupt transmission of Clas.

Acquisition, Replication and Inoculation of Candidatus Liberibacter asiaticus following Various Acquisition Periods on Huanglongbing-Infected Citrus by Nymphs and Adults of the Asian Citrus Psyllid.

The Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), is the primary vector of Candidatus Liberibacter asiaticus (Las) implicated as causative agent of citrus huanglongbing (citrus greening), currently the most serious citrus disease worldwide. Las is transmitted by D. citri in a persistent-circulative manner, but the question of replication of this bacterium in its psyllid vector has not been resolved. Thus, we studied the effects of the acquisition access period (AAP) by nymphs and adults of D. citri on Las acquisition, multiplication and inoculation/transmission. D. citri nymphs or adults (previously non-exposed to Las) were caged on Las-infected citrus plants for an AAP of 1, 7 or 14 days. These ‘Las-exposed’ psyllids were then transferred weekly to healthy citrus or orange jasmine plants, and sampled via quantitative polymerase chain reaction (qPCR) analysis 1–42 days post-first access to diseased plants (padp); all tested nymphs became adults 7–14 days padp. Our results indicate that following 1 or 7 day AAP as nymphs 49–59% of Las-exposed psyllids became Las-infected (qPCR-positive), whereas only 8–29% of the psyllids were infected following 1–14 day AAP as adults. Q-PCR analysis also indicated that Las titer in the Las-exposed psyllids (relative to that of the psyllid S20 ribosomal protein gene) was: 1) significantly higher, and increasing at a faster rate, following Las acquisition as nymphs compared to that following Las acquisition as adults; 2) higher as post-acquisition time of psyllids on healthy plants increased reaching a peak at 14–28 days padp for nymphs and 21–35 days padp for adults, with Las titer decreasing or fluctuating after that; 3) higher with longer AAP on infected plants, especially with acquisition as adults. Our results strongly suggest that Las multiplies in both nymphs and adults of D. citri but attains much higher levels in a shorter period of time post-acquisition when acquired by nymphs than when acquired by adults, and that adults may require longer access to infected plants compared to nymphs for Las to reach higher levels in the vector. However, under the conditions of our experiments, only D. citri that had access to infected plants as nymphs were able to inoculate Las into healthy citrus seedlings or excised leaves. The higher probability of Las inoculation into citrus by psyllids when they have acquired this bacterium from infected plants during the nymphal rather than the adult stage, as reported by us and others, has significant implications in the epidemiology and control of this economically important citrus disease.

Fourier transform infra-red spectroscopy using an attenuated total reflection probe to distinguish between Japanese larch, pine and citrus plants in healthy and diseased states

FTIR spectroscopy coupled with an Attenuated Total Reflection (ATR) sampling probe has been demonstrated as a technique for detecting disease in plants. Spectral differences were detected in Japanese Larch (Larix kaempferi) infected with Phytophthora ramorum at 3403cm-1 and 1730cm-1, from pine (Pinus spp.) infected with Bursaphelenchus xylophilus at 1070cm-1, 1425cm-1, 1621cm-1 and 3403cm-1 and from citrus (Citrus spp.) infected with ‘Candidatus liberibacter’ at 960cm-1, 1087cm-1, 1109cm-1, 1154cm-1, 1225cm-1, 1385cm-1, 1462cm-1, 1707cm-1, 2882cm-1, 2982cm-1 and 3650cm-1. A spectral marker in healthy citrus has been identified as Pentanone but is absent from the diseased sample spectra. This agrees with recent work by Aksenov, 2014. Additionally, the spectral signature of Cutin was identified in the spectra of Pinus spp. and Citrus spp. and is consistent with work by Dubis, 1999 and Heredia-Guerrero, 2014.

Transmission Rates of ‘Ca. Liberibacter asiaticus’ by Asian Citrus Psyllid Are Enhanced by the Presence and Developmental Stage of Citrus Flush.

Asian citrus psyllid (Diaphorina citri Kuwayama) transmits a bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) putatively responsible for a devastating citrus disease known as Asiatic huanglongbing (HLB) (citrus greening disease). The psyllid and disease have invaded many citrus-growing regions including the United States, where the disease is seriously jeopardizing the Florida citrus industry. We recently concluded research that showed CLas transmission rates are increased when citrus flush is present. Flush is any new leaf growth ranging in development from first emergence up until the leaves are fully expanded yet still tender. In an experiment with seedlings of a rootstock cultivar ‘US-942’, a 1-wk infestation of 20 Asian citrus psyllids from an infected colony resulted in 53–60% of seedlings becoming infected when flush was present compared with only 7% when no flush was present. In a second experiment with ‘US-942’, 77–97% of seedlings became infected when flush was present compared with 40% when no flush was present. A similar experiment with ‘Valencia’ sweet orange resulted in 23, 80, and 3% seedlings becoming infected when young, older, or no flush was present, respectively. Young plants are therefore more likely to contract HLB if flush is present, with older flush promoting higher infection rates under the conditions of this study. Based on this finding, healthy citrus should be protected from Asian citrus psyllid infestations throughout a flush. To evaluate germplasm for CLas resistance, inoculations using infected Asian citrus psyllid would best be achieved if flush is present.

Band Selection Using Forward Feature Selection Algorithm for Citrus Huanglongbing Disease Detection

, 2015Purpose: This study investigated different band selection methods to classify spectrally similar data - obtained from aerial images of healthy citrus canopies and citrus greening disease (Huanglongbing or HLB) infected canopies - using small differences without unmixing endmember components and therefore without the need for an endmember library. However, large number of hyperspectral bands has high redundancy which had to be reduced through band selection. The objective, therefore, was to first select the best set of bands and then detect citrus Huanglongbing infected canopies using these bands in aerial hyperspectral images. Methods: The forward feature selection algorithm (FFSA) was chosen for band selection. The selected bands were used for identifying HLB infected pixels using various classifiers such as K nearest neighbor (KNN), support vector machine (SVM), naive Bayesian classifier (NBC), and generalized local discriminant bases (LDB). All bands were also utilized to compare results. Results: It was determined that a few well-chosen bands yielded much better results than when all bands were chosen, and brought the classification results on par with standard hyperspectral classification techniques such as spectral angle mapper (SAM) and mixture tuned matched filtering (MTMF). Median detection accuracies ranged from 66-80%, which showed great potential toward rapid detection of the disease. Conclusions: Among the methods investigated, a support vector machine classifier combined with the forward feature selection algorithm yielded the best results.Keywords: Bayesian classification, Hyperspectral, K nearest neighbor, Multi-modal Bayesian classification, Support vector machine

Effect of host‐plant and infection with ‘Candidatus Liberibacter asiaticus’ on honeydew chemical composition of the Asian citrus psyllid, Diaphorina citri

AbstractThe Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), transmits the citrus greening pathogen ‘Candidatus Liberibacter asiaticus’ (CLas) by feeding on citrus phloem sap. Because phloem sap is rich in sugars but low in amino acids, ACP sucks large quantities and excretes most of it as honeydew. We studied the chemical composition of ACP honeydew on various host plants. Honeydew samples were analyzed with gas chromatography–mass spectrometry. Fourteen sugars, 13 amino acids, and six organic acids were detected in the honeydew of ACP. Sugars composed about 95% of the total compounds. Sucrose and trehalose were the predominant sugars, composing about 58 and 23% of the total sugars, respectively. Proline, asparagine, aspartic acid, and glutamic acid were the most abundant amino acids in ACP honeydew. The host plant and its infection with CLas had some effect on the honeydew composition. Glucose, chiro‐inositol, myo‐inositol, inositol, maltose, and turanose were lower in honeydew collected from CLas‐infected citrus compared to that collected from non‐infected trees. In CLas‐infected citrus (pineapple sweet orange, Citrus sinensis L. Osbeck) and Bergera koenigii (L.) Spreng. [curry leaf tree (both Rutaceae)] honeydews, valine, alanine, serine, glutamine, glycine, and the organic acids were lower than in honeydew from healthy citrus. Mannose, galactose, inositol, mannitol, an unknown disaccharide, and proline were higher in the honeydew collected from B. koenigii than in honeydew collected from healthy citrus (pineapple sweet orange), whereas fructose, chiro‐inositol, myo‐inositol, trehalose, and lactic acid were lower. The findings of this study help us understand the metabolism and the nutrient needs of ACP that transmits CLas, the pathogen of huanglongbing in citrus.

Movement ofDiaphorina citri(Hemiptera: Liviidae) Adults between Huanglongbing-Infected and Healthy Citrus

Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is the vector of huanglongbing (HLB or citrus greening disease). Preferences of D. citri adults differed for HLB-infected and healthy citrus under different maturity conditions. The presence or absence of young shoots had a significant influence on the choice by D. citri adults between HLB-infected and healthy citrus hosts. When citrus plants had young shoots, infected plants were more attractive than healthy ones to the adults. Also, D. citri adults fed for a longer time on infected plants with young shoots than on their healthy counterparts. In the absence of young shoots, D. citri adults were at first also more attracted to infected mature leaves, but after 38 h they turned to healthy mature leaves. In a multiple choice experiment, infected young shoots and healthy young shoots were the most attractive, followed by shoots with infected mature-yellow leaves and physiologically mature-yellow leaves, and lastly by healthy or infected mature-green leaves. In an experiment to measure the relative attractiveness of yellow, green, and white boards, yellow color boards attracted more adults than green and white boards, indicating that the adults preferred the yellow color. The results suggest that D. citri adults when first confronted with a choice are more attracted to infected citrus because of the color, but subsequently they move to healthy citrus perhaps because of either the poor nutrition or a feeding barrier in the infected hosts. This behavior appears to facilitate the pathogen’s spread.

Identification and Screening of Citrus Vein Phloem Degeneration (CVPD) on Brastagi Citrus Variety Brastepu (Citrus nobilis Brastepu) in North Sumatra Indonesia

Identification and screening of Citrus Vein Phloem Degeneration (CVPD) on Brastagi citrus variety Brastepu (Citrus nobilis Brastepu) in North Sumatra Indonesia is explained. The aim of the study is to explore the technique to obtain healthy Brastepu citrus to be used as a source of explants for in vitro propagation as a step in the preservation of threatened local citrus. The studies were conducted through collection of survived Brastepu citrus followed by screening the CVPD in the plants. Various procedures in the field and in the laboratory have been conducted to screen infected CVPD in Brastepu citrus. Visual examination from leaf, fruit, and seed appearance becomes preliminary information for CVPD infection in the citrus plant. Iodine test was used to support results obtained by visual observation. Histochemical test and the PCR analysis have confirmed that 12 samples of Brastepu citrus are free from CVPD and eight citrus are infected by CVPD. Adequate informations have been obtained to confirm healthy citrus tree which are then to be used as sources of explants for in vitro propagation in the preservation of Citrus nobilis Brastepu.

Ultrastructural Changes and Putative Phage Particles Observed in Sweet Orange Leaves Infected with 'Candidatus Liberibacter asiaticus'.

Huanglongbing (HLB), also known as citrus greening, is currently the most destructive citrus disease. Anatomical analyses of HLB-affected sweet orange were carried out by light and electron microscopy. As compared with healthy citrus, the phloem plasmodesmata were plugged with callose, and in some samples the phloem was collapsed. Chloroplast structures were deformed. Prophage sequences occupy a significant portion of the genome of 'Candidatus Liberibacter asiaticus' and have been used to distinguish strains from Yunnan and Guangdong provinces in China and Florida. Interestingly, a large number of possible putative phage particles were observed attached on the surface of 'Ca. L. asiaticus' cells in plants inoculated with strain FJ3 from Fujian Province, China. Phage particles have been observed previously only in periwinkle plants artificially inoculated in Florida with 'Ca. L. asiaticus' that carried the SC1-type prophage. PCR assays verified the presence of the SC1-type prophage sequences previously described from this bacterium in Florida in the FJ3 isolate. This is the first time that suspected phage particles have been observed in sweet orange trees infected with 'Ca. L. asiaticus.'

The Ability of the Antagonist Yeast Pichia Guilliermondii Strain Z1 to Suppress Green Mould Infection in Citrus Fruit.

In previous studies it was shown that Pichia guilliermondii strain Z1, isolated from healthy Moroccan citrus Valencia-Late oranges, was effective against Penicillium italicum. Here the effectiveness of strain Z1 was assessed against Penicillium digitatum, the causal agent of green mould, under different temperature (5-25°C) and relative humidity (RH) (45-100%) regimes for its reliable and large-scale application in packinghouse. All main effects and interactions were significant (P<0.0001). In the pathogen control, the largest lesion diameter was at an RH range between 98 and 100%, regardless of the incubation temperature. The efficacy of strain Z1 was not dependent on the environment and reduced disease incidence by >80%. Its applications as a formulated product significantly reduced the incidence of infected fruit (55%) and the percentage of infected wounds (47%) compared to the only pathogen control treatment. However, disease control with formulated product was significantly less than that obtained with thiabendazole (30%) or strain Z1 culturable cells (35%). These results highlight that strain Z1 is an effective biological control agent for control of green mould under varying environmental conditions, and control may be optimized by combining its use with other environmentally-safe post-harvest treatments or improved formulation.