Interactions between Indigenous Endophyte Bacillus subtilis L1-21 and Nutrients inside Citrus in Reducing Huanglongbing Pathogen Candidatus Liberibacter Asiaticus

Huanglongbing (HLB) pathogen Candidatus Liberibacter asiaticus (CLas) brings a great concern about the phloem nutrient transport in diseased plants. There is an urgent need to find the best management strategies to reduce the losses in the citrus industry worldwide. Endophytic bacteria are negatively affected by CLas pathogen, and these endophytes are associated with improved availability of nutrients and pathogen resistance. This study underpins the relationship between CLas pathogen, endophyte population and nutrients availability in citrus plants. The citrus plants were treated with Bacillus subtilis L1-21 and Hoagland solution to find out synergism efficacy to mitigate citrus HLB. We showed that citrus shoots in the presence of 50% Hoagland solution displayed maximum number of endophytes with 6.28 × 103 to 3.04 × 105 CFU/g. Among 50 candidate strains, B. subtilis L1-21 emerged as potential antagonist against surrogate strain Xanthomonas citri subsp. citri. The citrus half-leaf method identified that application of endophyte L1-21 with 50% Hoagland solution successfully reduces the CLas abundance. We point out that this combination results in a higher number of endophytes population with 2.52 × 104 to 9.11 × 106 CFU/g after 60 days, and reduces CLas pathogen abundance in asymptomatic HLB plants. In HLB symptomatic citrus plants, B. subtilis L1-21 potentially increases the endophyte population from 1.11 × 104 to 5.26 × 107 CFU/g in the presence of Hoagland solution, and pathogen abundance was reduced from 9.51 × 105 to 1.06 × 104 copies/g. Altogether, we suggested that the presence of endophyte L1-21 with Hoagland solution is more effective in HLB asymptomatic citrus plants, but a slight reduction of pathogen was observed in symptomatic plants. The findings revealed the role of indigenous citrus endophyte B. subtilis L1-21 along with other nutrients in the reduction of CLas pathogen abundance inside symptomatic and asymptomatic plants in citrus endophyte–nutrient–pathogen interplay.

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Tracing Penicillin Movement in Citrus Plants Using Fluorescence-Labeled Penicillin

Antibiotics ◽

10.3390/antibiotics8040262 ◽

2019 ◽

Vol 8 (4) ◽

pp. 262

Author(s):

Nabil Killiny ◽

Pedro Gonzalez-Blanco ◽

Yulica Santos-Ortega ◽

Fuad Al-Rimawi ◽

Amit Levy ◽

...

Keyword(s):

Vascular Tissue ◽

Vascular System ◽

Citrus Greening ◽

Application Site ◽

Asian Citrus Psyllid ◽

Diaphorina Citri ◽

Candidatus Liberibacter Asiaticus ◽

Citrus Industry ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus

Huánglóngbìng (HLB), citrus greening, is one of the most destructive diseases of citrus plants worldwide. In North America, HLB is caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus and is transmitted by the Asian citrus psyllid, Diaphorina citri. No cure exists at present, and the use of antibiotics for the control of HLB has gained interest due to the significant losses to the citrus industry. Because of unsatisfactory results when using foliar applications of antibiotics, concerns were raised regarding the uptake and translocation of these materials within trees. We, therefore, investigated a method that allows us to study the movement of antibiotic materials in citrus plants. Herein, we utilized a fluorescence-labeled penicillin, BOCILLIN™ FL-Penicillin (FL-penicillin), to study the uptake and translocation of penicillin in citrus plants. FL-penicillin was applied by puncture to the stem of young citrus seedlings and was traced by using fluorescence microscopy. After application, we detected FL-penicillin in the leaves and in the stem xylem and phloem tissues above and below the application site in both intact and partially bark-girdled citrus seedlings, indicating that it is easily taken up and transported through the plant vascular system. In addition, we detected FL-penicillin in the gut of D. citri, which were allowed to feed on the treated plants, suggesting translocation of this molecule into the vascular tissue. We propose that the use of fluorescent-labeled molecules could be an effective tool for understanding the uptake and translocation of antibiotics and other macromolecules in plants and insects.

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Development on Infected Citrus over Generations Increases Vector Infection by ‘Candidatus Liberibacter Asiaticus in Diaphorina citri’

Insects ◽

10.3390/insects11080469 ◽

2020 ◽

Vol 11 (8) ◽

pp. 469

Author(s):

Inaiara de Souza Pacheco ◽

Diogo Manzano Galdeano ◽

João Roberto Spotti Lopes ◽

Marcos Antonio Machado

Keyword(s):

Management Strategies ◽

Fourth Generation ◽

Diaphorina Citri ◽

Candidatus Liberibacter Asiaticus ◽

Candidatus Liberibacter ◽

Citrus Huanglongbing ◽

Liberibacter Asiaticus ◽

Citrus Orchards ◽

Vector Efficiency ◽

Control And Eradication

‘Candidatus Liberibacter asiaticus’ (CLas) is a major causal agent of citrus Huanglongbing (HLB), which is transmitted by Asian citrus psyllid (ACP), Diaphorina citri, causing severe losses in various regions of the world. Vector efficiency is higher when acquisition occurs by ACP immature stages and over longer feeding periods. In this context, our goal was to evaluate the progression of CLas population and infection rate over four ACP generations that continuously developed on infected citrus plants. We showed that the frequency of CLas-positive adult samples increased from 42% in the parental generation to 100% in the fourth generation developing on CLas-infected citrus. The bacterial population in the vector also increased over generations. This information reinforces the importance of HLB management strategies, such as vector control and eradication of diseased citrus trees, to avoid the development of CLas-infected ACP generations with higher bacterial loads and, likely, a higher probability of spreading the pathogen in citrus orchards.

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Understanding Citrus Greening Disease and Its Possible Management Strategies in Nepal

International Journal of Applied Sciences and Biotechnology ◽

10.3126/ijasbt.v9i4.40805 ◽

2021 ◽

Vol 9 (4) ◽

pp. 227-234

Author(s):

Sameer Pokhrel ◽

Swikriti Pandey ◽

Ashish Ghimire ◽

Savyata Kandel

Keyword(s):

Management Strategies ◽

Citrus Greening ◽

Insect Vector ◽

Candidatus Liberibacter Asiaticus ◽

Citrus Greening Disease ◽

The World ◽

Citrus Production ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Citrus Orchards

Huanglongbing (HLB), also known as citrus greening, is a devastating disease of citrus that has decimated several citrus orchards throughout the world. The disease is associated with three species of unculturable and phloem-limited bacteriae, Candidatus Liberibacter asiaticus, Candidatus Liberibacter africanus and Candidatus Liberibacter americanus. The most common species of bacteria found in Nepal is Candidatus Liberibacter asiaticus which is transmitted by an insect vector, Asian citrus psyllid (Diaphorina citri). This disease has been detected in several economically important citrus production areas of Nepal, which resulted in heavy yield loss. No cure for the disease has been discovered yet and it is essential to practice proper management strategies to maintain citrus health and sustain citrus production under HLB pressure. Several disease management approaches such as pathogen-free nursery establishment, use of disease tolerant rootstock cultivars, proper irrigation and nutrient supply, removal of HLB affected trees, and control of psyllid with frequent insecticide application are widely practiced throughout the world. This review article highlights the characteristics of the citrus greening disease and its insect vector and gives insights into their management techniques. Several technologically advanced options available to minimize the HLB infection might not be feasible currently in Nepal due to economic and topographic constraints. This article also aims to bring into focus the cost-effective methods that growers in Nepal can practice to mitigate the impact of HLB disease in their citrus orchards. Int. J. Appl. Sci. Biotechnol. Vol 9(4): 227-238.

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Genome Sequence Resource of ‘Candidatus Liberibacter asiaticus’ from Thailand

Plant Disease ◽

10.1094/pdis-07-19-1520-a ◽

2020 ◽

Vol 104 (3) ◽

pp. 624-626

Author(s):

Tao Li ◽

Narit Thaochan ◽

Jiaquan Huang ◽

Jianchi Chen ◽

Xiaoling Deng ◽

...

Keyword(s):

Genome Sequence ◽

Candidatus Liberibacter Asiaticus ◽

Citrus Industry ◽

Candidatus Liberibacter ◽

Citrus Huanglongbing ◽

Liberibacter Asiaticus

‘Candidatus Liberibacter asiaticus’ (Las) is an unculturable α-proteobacterium associated with citrus huanglongbing (HLB), a devastating disease currently threatening the citrus industry worldwide. Here, we present the genome sequence of Las strain TaiYZ2 from an HLB-affected pomelo tree in Hat Yai district, Songkhla Province, Thailand. The TaiYZ2 genome is composed of 1,230,623 bp with G+C content of 36.4%. This is the first Las genome sequence from Thailand, which will enrich current Las genome resource and facilitate HLB research and management.

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First report of ‘Candidatus Liberibacter asiaticus’ affecting sour orange in urban areas of Mayabeque, Cuba

10.1101/2020.03.25.008623 ◽

2020 ◽

Author(s):

Edel Pérez-López ◽

Tim J. Dumonceaux

Keyword(s):

Urban Areas ◽

Sour Orange ◽

Candidatus Liberibacter Asiaticus ◽

Citrus Industry ◽

Candidatus Liberibacter ◽

Citrus Huanglongbing ◽

Liberibacter Asiaticus ◽

Molecular Barcode ◽

Serious Disease ◽

First Time

Abstract‘Candidatus Liberibacter asiaticus’ (CLas) is an unculturable, Gram-negative, phloem restricted plant pathogenic bacterium associated with a very serious disease of citrus worldwide known as Citrus Huanglongbing (HLB). CLas is widely spread in the Americas. In Cuba, CLas has been associated with HLB symptoms and has seriously affected the Cuban citrus industry. In this short communication we discuss the identification of CLas-infected sour orange in urban areas of Mayabeque Province in Cuba, an area previously unexplored for the presence of HLB, and a host widely cultivated in gardens and yards along Cuba. We used for the first time the bacteria molecular barcode chaperonin-60 universal target (cpn60 UT) to identify and to detect CLas in HLB-symptomatic host plants.

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Restructuring citrus endophytic diversity through potential indigenous endophytes could eliminate huanglongbing pathogen Candidatus Liberibacter asiaticus

10.21203/rs.3.rs-74822/v1 ◽

2020 ◽

Author(s):

Shahzad Munir ◽

Yongmei Li ◽

Pengfei He ◽

Pengbo He ◽

Pengjie He ◽

...

Keyword(s):

Large Scale ◽

Pathogen Resistance ◽

Candidatus Liberibacter Asiaticus ◽

Citrus Grove ◽

Fold Reduction ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

The Impact ◽

Leaf Midrib

Abstract Background Huanglongbing (HLB) is a major botanical pandemic of citrus crops caused by Candidatus Liberibacter asiaticus (Clas). It is important to understand the different mechanisms involved in interaction of pathogen with plants to develop novel management strategy against HLB. However, until now there has been no control strategy to manage this disease in vitro and on large scale in citrus grove. We found that, indigenous endophyte Bacillus subtilis L1-21, a patented strain isolated from healthy citrus tree, may have the potential to reduce the impact of pathogen through restructuring of core endophytes. Results A novel half-leaf method was developed to test the efficacy of B. subtilis L1-21 against Clas. Concentration of B. subtilis L1-21 at 104 cfu ml− 1 resulted in a 1000-fold reduction in Clas copy densities per gram of leaf midrib (107 to 104) by 4 d after treatment. With endophytes, where HLB incidence was reduced to < 3% and Clas copy density was reduced from 109 to 104 pathogen g− 1 of diseased leaf midrib. We found that 16 of 93 tree samples became Clas-free and functional pathways and pathogen resistance genes were regulated in diseased citrus trees after treatment. Conclusions This is the first large-scale study using an indigenous endophyte and shows its potential utility in sustainable disease management through strengthening the citrus microbiome.

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Colonization of Dodder, Cuscuta indecora, by ‘Candidatus Liberibacter asiaticus’ and ‘Ca. L. americanus’

Phytopathology ◽

10.1094/phyto-100-8-0756 ◽

2010 ◽

Vol 100 (8) ◽

pp. 756-762 ◽

Cited By ~ 27

Author(s):

John S. Hartung ◽

Cristina Paul ◽

Diann Achor ◽

R. H. Brlansky

Keyword(s):

Cross Sections ◽

Quantitative Polymerase Chain Reaction ◽

Morphological Differentiation ◽

Ultrastructural Level ◽

Bacterial Cells ◽

Candidatus Liberibacter Asiaticus ◽

Citrus Industry ◽

Vessel Elements ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus

Huanglongbing, or citrus greening, threatens the global citrus industry. The presumptive pathogens, ‘Candidatus Liberibacter asiaticus’ and ‘Ca. L. americanus’ can be transferred from citrus to more easily studied experimental hosts by using holoparasitic dodder plants. However, the interaction between ‘Candidatus Liberibacter’ spp. and the dodder has not been studied. We combined quantitative polymerase chain reaction with electron microscopy to show that only 65% of tendrils of Cuscuta indecora grown on ‘Ca. Liberibacter’ spp.-infected host plants had detectable levels of the pathogen. Among tendrils that were colonized by Liberibacter in at least one 2 cm segment, most were not colonized in all segments. Furthermore, the estimated population levels of the pathogen present in serial 2 cm segments of dodder tendrils varied widely and without any consistent pattern. Thus, there was generally not a concentration gradient of the pathogen from the source plant towards the recipient and populations of the pathogen were sometimes found in the distal segments of the dodder plant but not in the proximal or middle segments. Populations of the pathogens ranged from 2 × 102 to 3.0 × 108 cells per 2 cm segment. On a fresh weight basis, populations as high as 1.4 × 1010 cells per g of tissue were observed demonstrating that ‘Ca. Liberibacter’ spp. multiplies well in Cuscuta indecora. However, 55% of individual stem segments did not contain detectable levels of the pathogen, consistent with a pattern of nonuniform colonization similar to that observed in the much more anatomically complex citrus tree. Colonization of dodder by the pathogen is also nonuniform at the ultrastructural level, with adjacent phloem vessel elements being completely full of the pathogen or free of the pathogen. We also observed bacteria in the phloem vessels that belonged to two distinct size classes based on the diameters of cross sections of cells. In other sections from the same tendrils we observed single bacterial cells that were apparently in the process of differentiating between the large and round forms to the long and thin forms (or vice versa). The process controlling this morphological differentiation of the pathogen is not known. The highly reduced and simplified anatomy of the dodder plant as well as its rapid growth rate compared with citrus, and the ability of the plant to support multiplication of the pathogen to high levels, makes it an interesting host plant for further studies of host–pathogen interactions.

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Detection of Citrus Huanglongbing-Associated ‘Candidatus Liberibacter asiaticus’ in Citrus and Diaphorina citri in Pakistan, Seasonal Variability, and Implications for Disease Management

Phytopathology ◽

10.1094/phyto-08-13-0224-r ◽

2014 ◽

Vol 104 (3) ◽

pp. 257-268 ◽

Cited By ~ 24

Author(s):

Muhammad F. Razi ◽

Manjunath L. Keremane ◽

Chandrika Ramadugu ◽

Mikeal Roose ◽

Iqrar A. Khan ◽

...

Keyword(s):

Seasonal Variability ◽

Management Strategies ◽

Diaphorina Citri ◽

Candidatus Liberibacter Asiaticus ◽

Term Survival ◽

Population Peak ◽

Average Maximum ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Maximum Temperatures

We report the detection of the huanglongbing (HLB)-associated bacterium ‘Candidatus Liberibacter asiaticus’ from both plants and insects in Pakistan and the seasonal variability in the numbers of ‘Ca. L. asiaticus’-positive psyllid vector, Diaphorina citri. Our studies showed that ‘Ca. L. asiaticus’ was detectable from trees in areas with maximum temperatures reaching nearly 50°C (average maximum of 42°C). However, the bacterium was present at very low levels in psyllids both in summer (June to August) and autumn (September to November) in contrast to reports from Florida, where the bacterium was detectable at very high levels during October to November. We hypothesize that hot summer temperatures in Pakistan may interfere with acquisition and replication of ‘Ca. L. asiaticus’ in psyllids and may lead to dead or nontransmissible ‘Ca. L. asiaticus’ in plants. Psyllid counts were very low in both summer and winter, showed a population peak (‘Ca. L. asiaticus’-positive vectors) in spring, and showed a larger peak (‘Ca. L. asiaticus’-free psyllids) in autumn. Natural thermotherapy during hot summers and a low vector population during environmental extremes may have played a major role in long-term survival of the citrus industry in Pakistan. These results may be useful in developing management strategies for U.S. citrus industries in Texas and California.

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Uptake, Translocation, and Stability of Oxytetracycline and Streptomycin in Citrus Plants

Antibiotics ◽

10.3390/antibiotics8040196 ◽

2019 ◽

Vol 8 (4) ◽

pp. 196 ◽

Cited By ~ 5

Author(s):

Fuad Al-Rimawi ◽

Faraj Hijaz ◽

Yasser Nehela ◽

Ozgur Batuman ◽

Nabil Killiny

Keyword(s):

Enzyme Linked Immunosorbent Assay ◽

Asian Citrus Psyllid ◽

Diaphorina Citri ◽

Candidatus Liberibacter Asiaticus ◽

In Planta ◽

Citrus Industry ◽

Candidatus Liberibacter ◽

Effective Program ◽

Liberibacter Asiaticus ◽

Root Tissues

Huanglongbing (HLB), or citrus greening, is the most destructive disease to the citrus industry. In Florida, it is caused by the bacterium, Candidatus Liberibacter asiaticus (CLas) and is transmitted by the Asian citrus psyllid, Diaphorina citri. Recent studies suggested that antibiotics could inhibit the growth of the CLas pathogen in planta. In the current study, we investigated the uptake and translocation of oxytetracycline and streptomycin in citrus seedlings. Oxytetracycline and streptomycin were delivered via root and stem and their level in various tissues was monitored using enzyme-linked immunosorbent assay (ELISA). Oxytetracycline and streptomycin were detected in the leaves, xylem, phloem, and root after root drench and stem delivery. High levels of antibiotics were detected in the roots after root drench, whereas high levels of antibiotics were detected in the canopy after stem delivery. The level of oxytetracycline detected in the phloem, xylem, and leaves after root drench was higher than that of streptomycin. Whereas the level of streptomycin in root was higher than that of oxytetracycline, indicating that streptomycin was bound to the xylem tissues. Oxytetracycline and streptomycin were detected in the phloem, xylem, leaves, and root tissues thirty-five days after the root incubation in 200 µg·mL−1 solution. These results demonstrated that oxytetracycline and streptomycin were relatively stable and could inhibit CLas growth for a couple of months in citrus trees. Observations reported in this study regarding the distribution and stability of oxytetracycline and streptomycin in citrus plants could be useful for designing an effective program for the control of HLB disease using antibiotics.

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Lack of Evidence for Transmission of ‘Candidatus Liberibacter asiaticus’ Through Citrus Seed Taken from Affected Fruit

Plant Disease ◽

10.1094/pdis-09-09-0595 ◽

2010 ◽

Vol 94 (10) ◽

pp. 1200-1205 ◽

Cited By ~ 22

Author(s):

John S. Hartung ◽

Susan E. Halbert ◽

Kirsten Pelz-Stelinski ◽

Ronald H. Brlansky ◽

Chunxian Chen ◽

...

Keyword(s):

Expressed Sequence Tag ◽

Zygotic Embryos ◽

Candidatus Liberibacter Asiaticus ◽

Repeated Testing ◽

Citrus Industry ◽

Candidatus Liberibacter ◽

Citrus Huanglongbing ◽

Liberibacter Asiaticus ◽

Citrus Seed ◽

Growth Habits

Citrus huanglongbing, putatively caused by the associated bacterium ‘Candidatus Liberibacter asiaticus’, is the greatest threat to the world citrus industry today. The bacterium is spread locally and regionally by the citrus psyllid Diaphorina citri, and also can be disseminated by propagation of contaminated scion budwood that is grafted to the appropriate rootstock. The planting of ‘Ca. Liberibacter asiaticus’-free trees is a component of a comprehensive strategy to manage huanglongbing. In contrast to the scion budwood, the rootstocks used to produce these trees are grown from seed. This research was undertaken to provide evidence as to whether or not ‘Ca. L. asiaticus’ can be transmitted through seed. Two groups of 360 or more seedlings each of various citrus species were grown from seed removed from fruit on trees that were symptomatic for huanglongbing and confirmed to be infected with ‘Ca. L. asiaticus’ by polymerase chain reaction (PCR) tests. These seedlings were tested multiple times over periods of up to 3 years. No symptoms typical of huanglongbing, such as blotchy leaf mottle, chlorotic shoots, or dieback of branches, were observed in these seedlings, and none of these 723 seedlings tested positive for the presence of ‘Ca. L. asiaticus’ even after repeated testing by sensitive quantitative PCR assays. Some sour orange seedlings did have quite pronounced and atypical growth, including stunting and mild to severe leaf malformation. These atypical growth habits were limited to seedlings that arose from zygotic embryos as determined by expressed-sequence tag simple-sequence repeat analyses. Thus, no evidence of transmission of ‘Ca. L. asiaticus’ via seed was obtained, and an earlier report of transmission of the pathogen through seed was not confirmed.

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