Reaction of some commercial citrus genotypes to Candidatus Liberibacter asiaticus in Iran

Reaction of some commercial citrus genotypes to Candidatus Liberibacter asiaticus in Iran | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Reaction of some commercial citrus genotypes to Candidatus Liberibacter asiaticus in Iran Shiva Safarpour Kapourchali, Mohammad Mehdi Faghihi, Nazanin Sadat Ebadi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8463450/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Citrus huanglongbing (HLB) associated with Candidatus Liberibacter spp. is the most destructive bacterial disease of citrus crops worldwide. In this study, the response of three-year-old seedlings of 14 commercial citrus cultivars and rootstocks to the Asian form of HLB disease ( Ca . L. asiaticus) was investigated through graft-inoculation. The seedlings' reactions to the HLB disease were evaluated based on the PCR detection of CLas after grafting, the disease incubation period (time between graft-inoculation and appearance of the first disease symptoms), and disease severity. Depending on the time of the positive PCR detection of CLas and appearance of disease symptoms, Mexican lime, Persian lime and Volkamer lemon were ranked at the highest level of tolerance (lowest susceptibility), and Rough lemon and Local orange were at the next level, i.e., semi-tolerance (intermediate susceptibility). Alemow, Bakraei, Rangpur lime, Cleopatra, Clementine mandarin, Local mandarin, Blood orange, and Washington navel orange were classified as susceptible to the disease, followed by Valencia orange, which was the most susceptible. In conclusion, all citrus genotypes studied, were affected by HLB. However, symptoms varied by cultivar, with the most severe observed on Valencia sweet orange and Local mandarin, while less severe symptoms were observed on Mexican lime, Persian lime and Volkamer lemon. Citrus cultivars Disease severity Huanglongbing Susceptibility Figures Figure 1 Introduction Huanglongbing (HLB), previously known as citrus greening, is the most devastating disease of citrus worldwide which is associated with three phloem limited Gram-negative α-proteobacteria, ‘ Candidatus Liberibacter asiaticus’ (CLas), ‘ Ca . L. africanus’ (CLaf), and ‘ Ca . L. americanus’ (CLam) (Bove, 2006). Both CLas and CLam are naturally transmitted through the Asian citrus psyllid, Diaphorina citri Kuwayama (Sternorrhyncha: Liviidae), while CLaf is transmitted by the African citrus psyllid Trioza erytreae Del Guercio (Sternorrhyncha: Triozidae). HLB-infected trees show different symptoms including blotchy mottle and corky vein in leaves, defoliation, yellowing and die back as well as small misshapen fruits, with color inversion (Bove 2006). The Asian form of HLB disease has been observed in citrus growing areas in southern Iran in Sistan-Baluchistan, Kerman, Hormozgan, and Fars provinces and the presence of CLas in HLB-affected citrus trees has been confirmed employing DNA-based methods (Alizadeh et al. 2010; Faghihi et al. 2009 ; Mohkami et al. 2011 ; Salehi et al. 2012 ; Salehi and Rasoulpour 2016 ). The disease is one of the causal agents of citrus decline in some areas of southern Iran, such as mandarin decline in Siyahoo in Hormozgan province of Iran (Faghihi 2018 ). Different strategies have been suggested in many citrus-producing countries with various results that have been indicated to reduce the development of HLB and help maintain tree productivity under field conditions. These measures include the use of insecticides and biological control to control psyllid vectors, improved foliar nutritional programs, the application of inorganic phosphorus (P) solution to the leaves, and the use of antibiotics like ampicillin, oxytetracycline, and streptomycin. Additionally, soil conditioners, plant defense inducers or activators, different graft/rootstock combinations, and thermotherapy may decrease the effects of HLB (Bassanezi et al. 2020 ). The development of citrus cultivars resistant to HLB is the most effective long-term control strategy for this damaging disease. Various research evaluated the effect of rootstock on the efficiency of citrus trees in the presence of HLB. Studies showed that the disease was transmitted to ‘Mosambi’ budded on Lisbon lemon, Bengal citron, Carrizo citrange, Troyer citrange, and trifoliate orange, but HLB symptoms stayed mild in these combinations. Hence, application of tolerant rootstocks was suggested as a part of an integrated control program (Bassanezi et al. 2020 ). Hajivand et al. ( 2011 ) stated that no HLB symptoms were observed in the mandarin scions when grafted on Citrus grandis rootstock with Citrus hystrix as an interstock. The high vigor-inducing rootstocks, particularly Volker lemon, may enable younger infected trees to survive longer than others (Albrecht et al. 2012). Citrus cultivars and hybrids have variable reactions to HLB disease. In general, orange ( Citrus sinensis ), tangerine ( C. reticulata ) and their hybrids are very sensitive, grapefruit ( C. paradisi ) and sour orange ( C. aurantium ) are semi-sensitive, and Poncirus trifoliata , Persian lime ( Citrus latifolia ), Eureka lemon ( Citrus limon ) and Mexican lime are tolerant to semi-tolerant to HLB disease (Chika et al. 2013 ; da Graca 1991; 2008; Deng et al. 2007 ; Folimonova et al. 2009 ). Some citrus species, such as Citrus indica and Citrus macroptera , did not develop disease symptoms under high inbreeding pressure and may be resistant to the disease (Bhagabati 1993 ). In Taiwan, the pomelo ( Citrus grandis ) cultivar was previously resistant to HLB, but finally became infected and showed HLB symptoms about 30 years after HLB first appeared (Huang and Chang 1980 ). Moreover, kumquat ( Furtunella margarita ) eventually became infected with HLB disease, exhibiting mottling and yellowing symptoms (Tsai et al. 2006 ). The final control of HLB is practical by identifying or producing resistant and tolerant citrus cultivars. The aim of this study was to investigate the reaction of three-year-old seedlings of commercial citrus cultivars and rootstocks in Iran to the causal agent of the Asian form of HLB disease ( Ca . L. asiaticus) through grafting infection. Materials and Methods Plant material and graft inoculation Experiments were conducted from September 2020 to September 2022 in a greenhouse where temperatures did not exceed 32°C. A number of commercial varieties of citrus in the country include Valencia sweet orange ( Citrus sinensis ), Common sweet orange ( C. sinensis ), Washington navel sweet orange ( C. sinensis ), Blood sweet orange ( C. sinensis ), Siyahoo mandarin ( Citrus reticulata ), Clementine mandarin ( C. reticulata ), Cleopatra mandarin ( Citrus reshni ), Mexican lime ( Citrus aurantifolia ), Bakraei ( Citrus spp.), Rangpur lime ( Citrus limonia ), Rough lemon ( Citrus jambhiri ), Macrophylla (Alemow) ( Citrus macrophylla ), Volkamer lemon ( Citrus volkameriana ) and Persian lime ( Citrus latifolia ) were prepared and six seedlings from each were kept under controlled conditions. Prior to graft-inoculation, all studied plants analyzed by polymerase chain reaction (PCR) with the primers A2/J5 for ‘ Ca. Liberibacter asiaticus/africanus’. The sources of inoculum were symptomatic branches of 3-year-old Valencia sweet orange seedlings which had previously been infected by CLas through graft-inoculation. Initial source of the infected grafts were mandarin trees severely affected by the Asian form of the HLB disease in an orchard in Siyahoo region (Hormozgan province, Iran). To evaluate citrus reactions to CLas infection, bark plus wood (budstick) in similar sizes (about 4 cm × 1 cm) were taken from symptomatic branches and used as sources of inoculum. Two budsticks were grafted onto the stem of each plant in two places on the middle part of the seedlings with 10 cm distance. The grafting tissues were firmly fixed by wrapping them with transparent plastic strips for a month. Furthermore, to maintain moisture and promote graft growth, the graft-inoculated part of the plants was kept in a plastic bag for about two weeks. Three seedlings for each genotype were grafted with healthy (HLB-free) scions as negative control. All plants were kept in an insect-free greenhouse at temperatures ranging from 32°C and 14°C (day and night, respectively) and around 12 to 14 hours of natural light (Lopes et al. 2008). PCR detection of CLas and disease incubation period The inoculated plants were inspected regularly for symptom expression of the disease and transmission success of CLas examined at two months intervals up to two years by conventional PCR and nested-PCR analysis of total DNA extracted from leaf samples. Based on the symptom appearance and the incubation period of the disease as well as PCR results, the response of each genotype was evaluated to HLB disease. The cultivars were ranked from the most sensitive to the most tolerant, based on the average number of days between the time of grafting and the date of the first detection of CLas in the grafted seedlings of each cultivar. A completely random design (CRD) with three replications (for each genotype) was used and data analysis was done by analysis of variance (ANOVA). Disease severity measurement Disease severity was evaluated based on symptoms of HLB on the graft-inoculated seedlings according to the rating scale proposed by Hajivand et al (2010) with brief modifications. Based on the leaf symptoms, the scale included: 0 = No symptom (Resistance), 1 = Mild (mottling and interveinal chlorosis symptoms), 2 = Moderate (Yellowing and/or Fe and Zn deficiencies like symptoms), 3 = Severe (defoliation and twigs dieback symptoms) Disease severity = Σ (a × b) / N × Z where Σ (a × b) = sum of the symptomatic plant and their corresponding rating, N = total number of sampled plants, and Z = highest rating. DNA Extraction and PCR assay DNA extraction was carried out according to the method of Hung et al., 1999 . In order to detection of CLas in the plant samples, the conventional PCR using the specific primers OI1/OI2c (Jagoueixet al. 1996 ) and a Nested-PCR using OI1/OI2c primer pair (ca. 1160 bp band) in the first round and CGO5R/CGO3F (Zhou et al., 2007 ) primer pair (ca. 800 bp band) in the second round were performed (Table 1 ). For both primer pairs, PCR was conducted in 35 cycles of 30 s at 94 °C (4 min at 95 °C for the initial denaturation), 40 s at 55 °C (60 °C for A2/J5 primer pair), and 80 s (1 min for A2/J5 primer pair) at 72 °C, and a final extension cycle of 10 min at 72 °C. The PCR reaction mixtures contained 10 µl of PCR Master Mix (Amplicon, Denmark), 0.4 pmol of each primer, and 2 µl of nucleic acid preparations. One microliter of diluted (1:10) PCR products from the first amplification was used as the template in the second round of PCR. DNA from healthy plants and sterile distilled water were used as the negative controls. Table 1 Characteristics of primers used in conventional and nested PCR tests to detect Ca. Liberibacter asiaticus Primer name Sequence Annealing (°C) Amplicon size (bp) Target Reference OI1 5′-GCGCGTATGCAATACGAGCGGCA-3′ 56 1160 16S rDNA Jagoueix et al. 1996 OI2c 5′-GCCTCGCGACTTCGCAACCCAT-3′ CGO3F 5′- RGGGAAAGATTTTATTGGAG-3′ 56 800 (nested-PCR) 16S rRNA Zhou et al. 2007 CGO5R 5′-GAAAATAYCATCTCTGATATCGT-3′ A2 5′- TATAAAGGTTGACCTTTCGAGTTT-3′ 60 669 (CLaf) β-operon Hocquellet et al. 1999 J5 5′- ACAAAAGCAGAAATAGCACGAACAA-3′ 703 (CLas) Electrophoresis of both conventional PCR and Nested PCR amplified fragments was performed using 8 µl of amplified fragments loaded on 1% agarose gel containing 2% safe stain (Sinnaclon, Iran). Afterward, the banding patterns of amplified fragments were observed using UV transilluminator. Then, based on the presence or absence of the expected band, positive and negative reactions were recorded in each experiment. Results Symptomatology and incubation period of the disease The inoculated plants were monitored in the greenhouse for two years, and different spectrums of disease symptoms were observed at various times post inoculation. These symptoms included mottling in the leaves, yellowing between the veins, symptoms similar to iron and zinc deficiency, yellow vein, and general yellowing of the plant (Fig. 1 ). Table 2 separately shows the symptoms in different hosts at intervals of three months. According to the Table 2 , no symptoms were observed in any of the commercial species and rootstocks of citrus until six months post inoculation (6 mpi). Eight and 9 mpi, disease symptoms appeared in one and two Valencia orange seedlings, respectively, while no symptoms were observed in other inoculated species like the control plants. About 12 mpi, the symptoms of the disease including mild mottling and yellowing between the veins in the upper leaves of the plant were observed in different cultivars of oranges and mandarins, Bakraei, Rangpur lime, and Alemow. However, no symptoms were observed in the seedlings of Mexican lime, Rough lemon, Volkamer lemon, and Persian lime 12 mpi. The symptoms of mild mottling and yellowing between the veins was observed during 16 to 19 mpi in Mexican lime, Rough lemon, Volkamer lemon, and Persian lime seedlings. Table 4 shows the incubation period of the disease after graft-inoculation in different citrus species. The incubation period of HLB disease was varied among the citrus genotypes. The comparison of the average incubation period between genotypes showed that the highest incubation periods were observed in Mexican lime, Volkamer lemon, Rough lemon, and Persian limes, which had a significant difference (p < 0.05) with other citrus genotypes. No significant difference (p < 0.05) was observed in incubation period of the disease between Local orange, Washington navel oranges, Blood oranges, Local mandarin, Clementine mandarin, Cleopatra mandarins, Bakraei, Rangpur lime, and Alemow (Table 4 ). ∗ These symptoms were observed in at least one of the three replicates inoculated for each genotype. No symptom (NS); Mild mottling (MM); Mottling (M); Interveinal chlorosis (IC); Fe and Zn deficiencies like symptoms (Fe/Zn Df); Yellowing in a part of the plants (PY); General yellowing (Y); Defoliation and die back (DD). Table 3 Analysis of variance related to the effect of citrus genotypes on incubation period of HLB disease. Source of variation S.V Degrees of freedom D.F Sums of squares S.S Mean of Square M.S F statistic F.S Coefficient of variation C.V% Genotype 13 342.0 26.3 30.7 ** 7.1 Error 28 24.0 0.8 - Total 41 366.0 - - ** Significant at 1% (p < 0.01) Table 4 Mean comparison of the incubation period of HLB disease in citrus genotypes. Citrus genotypes Incubation period (month) Valencia sweet orange 8.7 c* ± 0.33 Common sweet orange 11.7 b ± 0.33 Washington navel sweet orange 11.3 b ± 0.67 Blood sweet orange 11.3 b ± 0.33 Siyahoo mandarin 11.3 b ± 0.67 Clementine mandarin 12.0 b ± 0.58 Cleopatra mandarin 11.7 b ± 0.67 Mexican lime 18.0 a ± 0.58 Bakraei 11.3 b ± 0.33 Rangpur lime 11.7 b ± 0.33 Rough lemon 16.7 a ± 0.67 Alemow 11.7 b ± 0.67 Volkamer lemon 17.3 a ± 0.67 Persian lime 17.3 a ± 0.33 LSD 1.548 * Means with at least one similar letter had no significant difference based on LSD at 5% of probability level. PCR detection of Ca . L. asiaticus in the genotypes Table 6 shows the results of direct and nested-PCR tests in detection of Ca . L. asiaticus in the graft-inoculated plants at 60-day intervals. As can be seen, the sensitivity of the nested-PCR test in detecting CLas in the seedlings is higher than direct-PCR and could detect the infected plants at an earlier time. Statistical analysis and comparison of the average number of days between the time of graft-inoculation and the first time of positive detection of CLas by PCR in the seedlings showed that this period of time was significantly different between some citrus genotypes (Table 5 ). The average number of days until the first positive detection of CLas by PCR in citrus species showed that CLas was firstly detected in Valencia orange, i.e., 180 dpi (mean of direct and nested-PCR), and there was a significant difference ( p < 0.05) between Valencia orange with several species, including Local orange, Blood orange, Cleopatra mandarins, Rough lemons, Rangpur limes, Volkamer lemons, and Persian lime (Table 6 ). In the studied species, the disease agent was lastly detected in Mexican lime and Persian lime (on average 330 dpi), and Volkamer lemon (on average 300 dpi). Nevertheless, there was no significant difference ( p < 0.05) between Mexican lime, Persian limes, and Volkamer lemon in the number of days after inoculation until a positive detection in PCR (Table 6 ). Based on the average time between graft-inoculation and positive PCR detection of CLas in the inoculated species, it seems that Mexican lime, Persian lime, and Volkamer lemon had the most suppression against reproduction of CLas, because it was detected in these genotypes at a later time. Moreover, these genotypes had the highest incubation period of HLB as well as developed less severe HLB symptoms. Therefore, these genotypes are considered to be less susceptible to HLB than other citrus seedling verities. Rough lemon and Local orange were at the next level, that is, had the medium resistance to CLas. Alemow, Bakraei, Rangpur lime, Cleopatra mandarin, Clementine mandarin, Local mandarin, Blood oranges, and Washington navel orange were semi-sensitive, and Valencia orange was the most susceptible to CLas. Table 5 Analysis of variance of the individual and interaction effects of PCR method and citrus genotype on detection of Ca . Liberibacter asiaticus Source of variation Degrees of freedom Sums of squares Mean of Square F statistic Coefficient of variation PCR method 1 75.6 75.6 60.8** Genotype 13 158.2 12.2 9.79** 14.0 PCR method × Genotype 13 9.6 0.7 0.6ns Experimental Error 56 69.6 1.2 - Total 83 313.0 - - ** and ns: Significant and non-significant at 1%, respectively Table 6 Mean comparison of the number of days between grafting and HLB diagnosis times in different citrus cultivars Genotype First positive PCR detection of CLas (day post inoculation) Direct-PCR Nested-PCR Mean (Host) Valencia sweet orang †† 220 efg ± 20 140 h ± 20 † 180 E ± 22 Local sweet orange 300 abc ± 0 220 efg ± 20 260 BC ± 20 Washington navel sweet orange 240 def ± 35 200 fg ± 20 220 CDE ± 20 Blood sweet orange 280 bcd ± 20 200 fg ± 20 240 CD ± 22 Local mandarin (Siyahoo) 260 cde ± 40 180 gh ± 0 220 CDE ± 25 Clementine mandarin 260 cde ± 20 200 fg ± 20 230 CD ± 18 Cleopatra mandarin 280 bcd ± 20 200 fg ± 20 240 CD ± 22 Mexican lime 340 a ± 20 320 ab ± 20 330 A ± 13 Bakraei 240 def ± 0 180 gh ± 0 210 DE ± 13 Rangpur lime 280 bcd ± 20 200 fg ± 20 240 CD ± 22 Rough lemon 320 ab ± 20 260 cde ± 20 290 AB ± 18 Alemow 260 cde ± 20 200 fg ± 20 230 CD ± 18 Volkamer lemon 320 ab ± 20 280 bcd ± 20 300 AB ± 16 Persian lime 340 a ± 20 320 ab ± 20 330 A ± 14 PCR method mean † 281 A ± 8 221 B ± 9 A= Each number is the average of 3 numbers related to 3 grafted seedlings of each cultivar. † Means in each left column and below row having capital (individual effect of each factor) and †† the means in two middle columns having small letter (interaction of factors) with similar letters are not significantly different according to LSD test at p < 0.05. Table 7 Analysis of variance in relation to Disease severity progress of HLB in different citrus cultivars S.V D.F S.S M.S F.S Prob. Time 2 4.128 2.064 280.9** < 0.0001 Cultivar 13 1.054 0.081 11.04** < 0.0001 Time × Cultivar 26 0.242 0.009 1.26ns 0.2100 Error 84 0.617 0.007 - - Total 125 6.041 - - - ns, *,** Not Significant, Significant at p < 0.05 and p < 0.01, respectively. Table 8 Disease severity progress of HLB in different citrus cultivars Genotype Disease severity Mean of Genotype 12 months 18 months 24 months Valencia sweet orange Ψ 0.3333 de ± 0.0000 0.5555 bc ± 0.0641 0.7777 a ± 0.0000 † 0.5555 A ± 0.0668 Local sweet orange 0.2222 ef ± 0.0641 0.4444 cd ± 0.0641 0.5555 bc ± 0.0641 0.4074 BC ± 0.0586 Washington navel sweet orange 0.2222 ef ± 0.0641 0.4444 cd ± 0.0641 0.5555 bc ± 0.0641 0.4074 BC ± 0.0586 Blood sweet orange 0.2222 ef ± 0.0641 0.4444 cd ± 0.0641 0.6666 ab ± 0.0641 0.4074 BC ± 0.0586 Local mandarin (Siyahoo) 0.3333 de ± 0.0000 0.5555 bc ± 0.0641 0.7777 a ± 0.0000 0.5555 A ± 0.0668 Clementine mandarin 0.2222 ef ± 0.0641 0.4444 cd ± 0.0641 0.6666 ab ± 0.0641 0.4444 B ± 0.0717 Cleopatra mandarin 0.1111 fg ± 0.0000 0.4444 cd ± 0.0641 0.6666 ab ± 0.0000 0.4074 BC ± 0.0828 Mexican lime 0.0000 g ± 0.0000 0.2222 ef ± 0.0000 0.4444 cd ± 0.0641 0.2222 F ± 0.0668 Bakraei 0.2222 ef ± 0.0641 0.4444 cd ± 0.0641 0.5555 bc ± 0.0000 0.4074 BC ± 0.0556 Rangpur lime 0.1111 fg ± 0.0000 0.4444 cd ± 0.0641 0.5555 bc ± 0.0641 0.3703 BCD ± 0.0717 Rough lemon 0.0000 g ± 0.0000 0.4444 cd ± 0.0641 0.5555 bc ± 0.0641 0.3333 CDE ± 0.0888 Alemow 0.1111 fg ± 0.0000 0.4444 cd ± 0.0641 0.6666 ab ± 0.0000 0.4074 BC ± 0.0828 Volkamer lemon 0.0000 g ± 0.0000 0.4444 cd ± 0.0641 0.4444 cd ± 0.0000 0.2963 DEF ± 0.0764 Persian lime 0.0000 g ± 0.0000 0.3333 de ± 0.0000 0.4444 cd ± 0.0641 0.2592 EF ± 0.0693 Mean of Time † 0.1508 C ± 0.0200 0.4365 B ± 0.0179 0.5872 A ± 0.0198 † Means having capital in the right column and below row with same letters are not significantly difference according to LSD test (p < 0.05). Ψ Means having small letter in middle columns with same letters are not significantly difference according to LSD test (p < 0.05). Disease severity Based on the disease symptoms, the severity of HLB disease was evaluated in different citrus hosts at 12, 18 and 24 months after CLas graft-inoculation (Table 7 ). As shown in Table 8 , 12 months post inoculation, HLB symptoms were not observed on Mexican lime, Rough lemon, Volkamer lemon and Persian lime, but mild HLB symptoms were observed on other hosts. The disease severity in the hosts increased over time and the most disease severity for each genotype was observed at 24 months post inoculation. After 24 months of inoculation, the lowest disease severity (0.44, moderate symptoms) was observed in Mexican lime, Volkamer lemon and Persian lime, and the highest disease severity (0.77, severe symptoms) was observed in Valencia orange and Local mandarin (Siyahoo). Discussion The Asian form of citrus HLB caused by Candidatus Liberibacter asiaticus was first found in Iran in 2008 (Faghihi et al., 2009 ). The disease is transmitted by the Asian citrus psyllid, Diaphorina citri , which is widespread in citrus growing areas in southern Iran (Alizadeh et al. 2022; Salehi et al. 2012 ). In this research, the reaction of three-year-old seedlings of 14 commercial cultivars and rootstocks of citrus to the causal agent of the Asian form of HLB disease ( Ca . L. asiaticus) was investigated through graft-inoculation. Based on the PCR detection of CLas after grafting, the disease incubation period (the time between graft-inoculation and the appearance of the first symptoms of the disease), and disease severity in the seedlings, their reaction to the HLB disease were evaluated. In pathogen transmission through grafting, the primary inoculum present in the tissue of the scion is directly transferred to the tissue of the host plant. In this method, unlike the transmission with psyllid and Cuscuta spp., the exact time of inoculation, the amount of initial inoculum (the number and size of the scions), and the inoculation location can be controlled to a large extent (Batool et al. 2007 ). However, there are limitations in this transmission method, such as the survival of the scion, and the degree of compatibility of the scion tissue with the rootstock tissue. The symptoms observed in the infected citrus seedlings compared with the negative PCR seedlings (negative control) and the type of the symptoms in the inoculated plants was regularly recorded. Besides that, the PCR results confirmed the CLas infection in the seedlings. The symptoms of HLB disease are not very specific and the type and severity of the symptoms occurred in the greenhouse may be different from the symptoms in nature (due to the age of the trees, climatic conditions, and the presence of the vector). In nature, typical symptoms of the disease include yellowing shoots, blotchy mottle of leaves, corky veins, and localized symptoms of HLB spreads over the canopy, finally leading to defoliation and dieback. Moreover, the infected plants usually produce small, asymmetrical, bitter-tasting fruits with color inversion (Bove 2006). However, in our greenhouse experiment, only some of the symptoms were observed, and in many cases, the predominant symptoms in the infected seedlings were mottling, general yellowing, interveinal yellowing, and symptoms resembling iron and zinc deficiencies. The chlorotic pattern's symptoms similar to those of zinc and iron deficiencies can be seen in other diseases such as citrus tristeza, citrus stubborn, and phytoplasma infection. In the studied citrus genotypes, the symptoms of the disease appeared between eight and 18 months on different seedlings, and the incubation period varied between seedlings up to 10 months. On the other hands, the CLas could be detected by PCR in the inoculated plants several months before the appearance of the disease symptoms (Table 6 ). For example, the first disease symptoms were noticed on Valencia orange (the most susceptible cultivar) at 8 to 9 mpi, while the first positive detection of CLas was found in Valencia orange seedlings by the nested-PCR method at 4 to 6 mpi and by the direct-PCR assay at 6 to 8 mpi. Therefore, on average, 2 to 4 months before the appearance of HLB symptom, the CLas was detected in inoculated Valencia orange seedlings by direct- and nested-PCR tests, respectively. Disease symptoms were appeared in other genotypes, such as another cultivar of oranges and tangerines, Bakraei, Rangpur lime, and Alemow, between 10 and 13 mpi. In these genotypes, the first positive PCR detection of CLas occurred between 6 and 8 mpi, or approximately 4 to 5 months prior to the onset of symptoms. The longest incubation period of the HLB disease was recorded between 16 and 19 mpi in the seedlings of Mexican lime, Rough lemon, Volkamer lemon, and Persian lime, while PCR confirmed that these genotypes were infected with CLas between 10 and 12 mpi. Hence, even in more tolerant plants, the CLas could be detected by PCR up to 12 mpi and several months before the appearance of the disease symptoms. It is difficult to detect CLas prior to the symptom expression, especially under natural conditions, due to its non-uniform distribution and low concentration in infected citrus trees. The highly sensitive nested-PCR is an appropriate approach for detecting CLas during the disease's incubation period and can reveal the presence of HLB infection several months before direct-PCR. Molecular methods such as PCR have been used successfully to identify and detect the different species of Ca . Liberibacter (Jagoueixet al. 1996 ; Zhou et al. 2007 ; Hocquellet et al. 1999 ). In some cases, direct-PCR may fail to detect the CLas in infected trees due to the low concentration of the bacterium or present of disturbing metabolites in the tissue extract. In this study, in addition to the direct-PCR, a sensitive nested-PCR was performed to detect the CLas. Overall, there was a significant difference between the nested-PCR and direct-PCR methods for detecting CLas in the inoculated plants. As expected, the nested-PCR had the higher sensitivity, and CLas was detected in the inoculated plants at least two months before direct-PCR. However, the false positive results may occur, especially in nested-PCR assay, hence DNA from a healthy plant and sterile distilled water should be used in each reaction as negative controls. The development of disease symptoms and the rate of CLas reproduction in host plant tissue reveal the different levels of sensitivity and tolerance of the host plant. Therefore, the faster the disease agent (CLas) can settle in the host and multiply, the sooner the pathogen is detected by the PCR method in the inoculated plants, and the earlier the disease symptoms appear, indicating that the host plant is more susceptible to the disease. The detection time of the bacterium in the phloem of the host plant, along with the appearance time of the disease symptoms, could be an appropriate relative index to evaluate the response of different citrus cultivars to the pathogen and compare them with each other. Thus, an earlier detection of CLas in some hosts, such as Valencia orange, indicates better reproduction and a faster increase in the concentration of the bacterium until reaching the detecting threshold by PCR. Furthermore, a correlation was found between the time of the positive PCR detection of CLas after graft inoculation, disease incubation period, and disease severity in the examined cultivars and rootstocks. It means that the more sensitive inoculated seedlings became rapidly positive in PCR due to the higher concentration of the pathogen, and the symptoms of the disease appeared earlier (shorter incubation period) and disease severity was higher. The plant hosts with long disease incubation period and mild symptoms were considered less susceptible to HLB disease. According to the investigations, the concentration of the pathogenic bacterium in the plant is not uniform, and it is higher in the symptomatic parts of the infected plant. This demonstrates the enhanced proliferation of the bacterium in the symptomatic parts of the plant (Kunta and de Graca 2014). Based on the time of the positive PCR detection of CLas and the symptom appearance of the disease, Mexican lime, Persian lime, and Volkamer lemon placed at the highest tolerance level (lowest susceptibility), and Rough lemon and Local orange were at the next level, i.e., semi-tolerance (medium susceptibility). The next group included Alemow, Bakraei, Rangpur lime, Cleopatra mandarin, Clementine mandarin, Local mandarin, Blood orange, and Washington navel orange, which were classified as susceptible to the disease, and the last group included the Valencia orange, which was the most susceptible to the disease. In the surveys in the citrus growing areas in southern Iran, it has been observed that the Asian citrus psyllid ( Diaphorina citri ) has a host preference for Mexican lime and establishes a larger population on it, but the lower severity of the HLB disease has been found on Mexican lime trees. Under natural conditions, the incidence and severity of HLB are considerably higher in orange and mandarin trees than in other citrus species, despite the fact that D. citri populations on these trees are much smaller than those on Mexican lime. Several studies have shown that orange and tangerine cultivars are sensitive, grapefruits and sour orange are semi-sensitive, and lemons are more tolerant to the HLB disease (Folimonova et al. 2009 ; Halbert and Manjunath 2004 ; Miyakawa and Zhao 1990 ). Wild genotypes of citrus, such as Citrus indica and Citrus macroptera in Northeast India, remained asymptomatic despite infected psyllid in the region (Bhagabati 1993 ). Poncirus trees and seedlings ( Poncirus trifoliata ) are also very resistant to this disease (Miyakawa 1980 ). Mexican lime had relatively high tolerance against the disease, and uses as a rootstock in different parts of Iran. Persian lime is tolerant to both HLB and lime witches’ broom, making it suitable for planting in locations such as southern Iran where both diseases prevail. There have also been reports of the relative resistance of several rootstocks to this disease. In conclusion, all the studied citrus genotypes, irrespective of their rootstock, were susceptible to the HLB disease. However, symptoms vary from cultivar to cultivar, with the most severe found on Valencia sweet orange, and Local mandarin. Symptoms were less severe on Mexican lime, Persian lime and Volkamer lemon. The studies suggest that proper tolerance to HLB, with reduced symptoms and probably reduced CLas titer might be usually found in rootstock cultivars and symptomless natural biotypes. These sources can be used as rootstocks, and for breeders to produce resistant cultivars. Due to the lack of natural resistance in citrus species, employing genetic engineering to create HLB-resistant commercial cultivars would have a high chance of success (Bove 2006). Over the last few years, research has focused on genome-editing technologies such as CRISPR/Cas9 and single-guide RNA (sgRNA) to enhance crop development. CRISPR/Cas9 is a specific and straightforward technique used to modify the genome of crops to produce disease-resistant/tolerant plants, and it might be the most accepted approach for crop improvement in the future (Ghosh et al. 2023 ). Further work is needed to assess investigating the response of more citrus genotypes to CLas. Furthermore, in the citrus genotypes, CLas concentration and multiplication process can be checked in different organs especially root system and compared with each other using quantitative real time-PCR. Declarations Author Contribution Shiva Safarpour Kapourchali : Writing – original draft, Methodology, Investigation. Mohammad Mehdi Faghihi : Writing – review & editing, Conceptualization, Methodology, Investigation, Formal analysis. Nazanin Sadat Ebadi: Methodology, Investigation Ali Alizadeh Aliabadi : Writing – review & editing. Hamed Hasanzadeh Khankahdani: Software, Methodology, Formal analysis. All the authors have read and agreed to the published version of the manuscript. Data availability All data generated or analysed during this study are included in this published article [and its supplementary information files]. Acknowledgements The authors would like to thank Islamic Azad University of Iran, Varamin-Pishva Branch, and Fars Agricultural and Natural Resources Research and Education Center for supporting this work. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Albrecht U, Hall DG, Bowman KD (2014) Transmission efficiency of ‘ Candidatus Liberibacter asiaticus’ and progression of huanglongbing disease in graft and psyllid-inoculated citrus. 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Phytopathology 96:23 Chen J, Pu X, Deng X, Liu S, Li H, Civerolo E (2009) A phytoplasma related to Candidatus Phytoplasma asteris detected in citrus showing huanglongbing (yellow shoot disease) symptoms in Guangdong, P.R. China. Phytopathology 99:236–242 Chika C, Nwugo, Yongping D, Hong L (2013) Study on Citrus Response to Huanglongbing Highlights a Down-Regulation of Defense-Related Proteins in Lemon Plants Upon ‘ Ca . Liberibacter asiaticus’ Infection. PLoS ONE 8:e67442 da Graça JV (1991) Citrus greening disease. Annu Rev Phytopathol 29:109–136 da Graça JV (2008) Biology, history and world status of Huanglongbing. Texas A & M University-Kingsville, Citrus Center, Weslaco TX 78596, USA De Barro PJ, Sherratt TN, Brookes CP, David O, Maclean N (1995) Spatial and temporal variation in British field populations of the grain aphid Sitobion avenae (F.) (Hemiptera: Aphididae) studied using RAPD-PCR. Proc R Soc Lon Ser-B 262:321–327 Deng S, Hiruki C (1991) Amplification of 16S rRNA genes from culturable and nonculturable mollicutes. J Microbiol Meth 14:53–61 Deng X, Zhou G, Li H (2007) Nested-PCR detection and sequence confirmation of ‘ Candidatus Liberibacter asiaticus’ from Murraya paniculata in Guangdong, China. Plant Dis 91:1051 Ding F, Wang G, Yi G, Zhong Y, Zeng J, Zhou B (2005) Infection of Wampee and Lemon by the citrus Huanglongbing pathogen ( Candidatus Liberibacter asiaticus) in China. J Plant Pathol 87:207–212 Faghihi MM, Salehi M, Bagheri A, Izadpanah K (2009) First report of citrus huanglongbing disease on orange in Iran. Plant Pathol 58:793 Faghihi MM, Taghavi SM, Salehi M, Golmohammadi M (2016) First report of huanglongbing disease on Mexican lime in Iran. In: Proceedings of the 20th International Organization of Citrus Virologist (IOCV) conference, April 10–15, 2016, Chongqing, China.p.45 Faghihi MM (2018) Investigation of association of phloem-limited prokaryotes with Siyahoo tangerine trees showing misshapen and inverted color change symptoms in fruit. 23rd Iranian Plant Protection Congress. 27–30 August 2018, Gorgan. IRAN. Feng YC, Huang TH, Su HJ (2015) Detection and inoculation of peanut witches' broom phytoplasma (16SrII-A) and periwinkle leaf yellowing Phytoplasma (16SrI-B) in citrus cultivars in Taiwan. J Phytopathol 163:364–376 Folimonova SY, Robertson CJ, Garnsey SM, Gowda S, Dawson WO (2009) Examination of the responses of different genotypes of citrus to Huanglongbing (citrus greening) under different conditions. Phytopathology 99:1346–1354 Golmohammadi M, Banihashemian SM, Raheb S, Alian YM, Faghihi MM, Salehi M (2016) First report of Candidatus Liberibacter asiaticus associated with HLB disease in grapefruit from south of Iran. In: Proceedings of the 20th International Organization of Citrus Virologist (IOCV) conference, April 10–15, 2016, Chongqing, China.p.48 Ghosh D, Kokane S, Savita BK, Kumar P, Sharma AK, Ozcan A, Kokane A, Santra S (2023) Huanglongbing pandemic: Current challenges and emerging management strategies. Plants 12:160. https://doi.org/10.3390/plants12010160 Hajivand S, Thohirah Lee A, Kamaruzaman S, Siti Nor AA, Nur Ashikin Psyquay A (2009) Differential reaction of citrus species in Malaysia to huanglongbing (HLB) disease using grafting method. Am J Agric Biol Sci 4:32–38 Hajivand S, Thohirah Lee A, Kamaruzaman S, Siti Nor AA (2011) Potential use of selected citrus rootstocks and interstocks against HLB disease in Malaysia. Crop Prot 30:521–525 Halbert SE, Manjunath KL (2004) Asian citrus psyllids (Sternorrhyncha: Psyllidae) and greening disease of citrus: A literature review and assessment of risk in Florida. Fla Entomol 87:330–353 Hocquellet A, Bové JM, Garnier M (1999) Isolation of DNA from the uncultured Candidatus Liberobacter species associated with citrus Huanglongbing by RAPD. Curr Microbiol 38:176–182 Huang CH, Chang CA (1980) Studies on the relation of mycoplasma-like organism with the decline of Wentan pummelo in Taiwan. J Agricul Res China 29:13–19 Hung TH, Wu ML, Hong-J S (1999) Development of a rapid method for the diagnosis of citrus greening disease using the polymerase chain reaction. J Phytopathol 147:599–604 Jagoueix S, Bové JM, Garnier M (1996) PCR detection of the two ‘ Candidatus ’ Liberibacter species associated with greening disease of citrus. Mol Cell Probes 10:43–50 Kunta M, da Graça JV, Malik NS, Louzada ES, Setamou M (2014) Quantitative distribution of Candidatus Liberibacter asiaticus in the aerial parts of the huanglongbing-infected citrus trees in Texas. Hort Sci 49(1):65–68 Lee I-M, Bertaccini A, Vibio M, Gundersen DE (1995) Detection of multiple phytoplasmas in perennial fruit trees with decline symptoms in Italy. Phytopathology 85:728–735 Lopes A, Frare GF (2008) Graft transmission and cultivar reaction of citrus to ‘ Candidatus Liberibacter americanus. Plant Dis 92:21–24 Miyakawa T (1980) Experimentally-induced symptoms and host range of citrus likubin (greening disease). Ann Phytopath Soc Japan 46:224–230 Miyakawa T, Tsuno K (1989) Occurrence of citrus greening in the southern islands of Japan. Ann Phytopath Soc Japan 66:667–670 Miyakawa T, Zhao XY (1990) Citrus host range of greening disease, pp. 118–121. In: Aubert, B., S. Tontyaporn, and D. Buangsuwon (eds.). Proc. 4th International Asia Pacific Conference on Citrus Rehabilitation. FAO-UNDP Mohkami A, Sattari R, Lori Z, Ehsani A, Nazemi A (2011) First report of citrus huanglongbing in the Orzooiyeh region in Kerman province (Orzooiyeh). Iran J Plant Pathol 47:105 Roistacher CN (1991) Graft-transmissible diseases of citrus. Handbook for detection and diagnosis. FAO, Rome, p 286 Rouse RE, Ozores-Hampton M, Roka FM, Roberts P (2017) Rehabilitation of huanglongbing affected citrus trees using severe pruning and enhanced foliar nutritional treatments. Hort Sci 52:972–978 Ruangwong O, Akarapisan A (2006) Detection of Candidatus Liberibacter asiaticus causing Citrus Huanglongbing disease. J Agricul Technol 2:111–120 Salehi M, Rasoulpour R (2016) First report of 'Candidatus Liberibacter asiaticus' associated with Hunglongbing in Fars province, Iran. J Plant Pathol 51(4):563–566 Salehi M, Faghihi MM, Khanchezar A, Bagheri A, Izadpanah K (2012) Distributioin of citrus huanglongbing disease and its vector in southern Iran. Iran J Plant Pathol 48(2):195–208 Schneider B, Seemüller E, Smart CD, Kirkpatrick BC (1995) Phylogenetic classification of plant pathogenic mycoplasma-like organisms or phytoplasmas. In Molecular and Diagnostic Procedures in Mycoplasmology 2:369–380. Edited by S. Razin & J. G. Tully. San Diego: Academic Press Sechler A, Schuenzel EL, Cooke P, Donnua S, Thaveechai N, Postnikova E, Stone AL, Schneider WL, Damsteegt VD, Schaad NW (2009) Cultivation of ‘ Candidatus Liberibacter asiaticus’,‘ Ca. L. africanus’, and ‘ Ca. L. americanus’ associated with huanglongbing. Phytopathology 99:480–486 Tsai CH, Su HJ, Liao YC, Hung TH (2006) First report of the causal agent of huanglongbing ( Candidatus Liberibacter asiaticus) infecting kumquat in Taiwan. Plant Dis 90:1360 Villechanoux S, Garnier M, Renaudin J, Bovè JM (1993) The genome of the non-cultured bacterial-like organism associated with citrus greening disease contains the nusGrplKAJL-rpoBC gene cluster and the gene for a bacteriophage DNA polymerase. Curr Microbiol 26:161–166 Zhou LJ, Gabriel DW, Duan YP, Halbert SE, Dixon WN (2007) First report of dodder transmission of huanglongbing from naturally infected Murraya paniculata to citrus. Plant Dis 91:227 Table 2 Table 2 is available in the Supplementary Files section. Supplementary Files Table2.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8463450","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":594489730,"identity":"bec6e15e-dc2d-4bc2-b071-d78c17c81c19","order_by":0,"name":"Shiva Safarpour Kapourchali","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shiva","middleName":"Safarpour","lastName":"Kapourchali","suffix":""},{"id":594489731,"identity":"acdf9f2d-a396-4493-b458-17f5e7a573de","order_by":1,"name":"Mohammad Mehdi Faghihi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYJCCDwwMB3gMmJkPMDA2EKeDcQZEC1sCaVoYDBh4DIjTojvtAGPDj193ZMzZeb5J/NxhI8fAfvjoBnxazG4nMDb29j3jsWzm3SbZeybNmIEnLe0GAS3sD3h7DvMYHObdJsHbdjixQYLHjJAWxsa/YC08zyT/EqulmecHWAubNJG2JDY2yzaAtLAZW8u2pRmzEfZL8sHGN38O2xucP/zw5ts2Gzl+9sPH8GphAMUFYxuYxSIBItnwK4eBP2CS+QNxqkfBKBgFo2CkAQDC0lBhPcS6BQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-5147-1844","institution":"Fars Agricultural and Natural Resources Research and Education Center","correspondingAuthor":true,"prefix":"","firstName":"Mohammad","middleName":"Mehdi","lastName":"Faghihi","suffix":""},{"id":594489732,"identity":"2488a34f-e4ea-43c5-891b-7c19bf884f26","order_by":2,"name":"Nazanin Sadat Ebadi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Nazanin","middleName":"Sadat","lastName":"Ebadi","suffix":""},{"id":594489733,"identity":"5749644c-9d97-4308-8984-03f705a1195f","order_by":3,"name":"Ali Alizadeh Aliabadi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"Alizadeh","lastName":"Aliabadi","suffix":""},{"id":594489734,"identity":"363617b7-f17a-490a-b18b-d92985ee84cb","order_by":4,"name":"Hamed Hasanzadeh Khankahdani","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hamed","middleName":"Hasanzadeh","lastName":"Khankahdani","suffix":""}],"badges":[],"createdAt":"2025-12-28 01:03:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8463450/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8463450/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103339884,"identity":"ac75ce0d-4bfb-4611-8f10-ea45d076cc40","added_by":"auto","created_at":"2026-02-24 15:16:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1901681,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSymptoms of HLB in some graft-inoculated plants in greenhouse. a: Mottling and interveinal chlorosis in Local orange (12 months post inoculation= 12 mpi); b:Mottling in Valencia orange leaves (9 mpi); c: Interveinal chlorosis and mottling in local mandarin leaves (12 mpi); d: Yellowing and Fe deficiency like symptoms in Mexican lime (24 mpi); e: mottling and Fe deficiency like symptoms in Persian lime (18 mpi); f: Yellowing and Fe deficiency like symptoms in Bakraei (12 mpi).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8463450/v1/6cccadec79ada29740925ac3.png"},{"id":108807555,"identity":"62aca9e1-ca14-4db9-8782-88338ad3a22f","added_by":"auto","created_at":"2026-05-08 15:30:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3775929,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8463450/v1/f7b64965-ad4a-427a-b774-abf489728f77.pdf"},{"id":103339883,"identity":"97f70c19-f232-4615-b19e-03fe8d778d12","added_by":"auto","created_at":"2026-02-24 15:16:10","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18440,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8463450/v1/264cb39b22accf75ce4976d3.docx"}],"financialInterests":"","formattedTitle":"Reaction of some commercial citrus genotypes to Candidatus Liberibacter asiaticus in Iran","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHuanglongbing (HLB), previously known as citrus greening, is the most devastating disease of citrus worldwide which is associated with three phloem limited Gram-negative α-proteobacteria, \u0026lsquo;\u003cem\u003eCandidatus\u003c/em\u003e Liberibacter asiaticus\u0026rsquo; (CLas), \u0026lsquo;\u003cem\u003eCa\u003c/em\u003e. L. africanus\u0026rsquo; (CLaf), and \u0026lsquo;\u003cem\u003eCa\u003c/em\u003e. L. americanus\u0026rsquo; (CLam) (Bove, 2006). Both CLas and CLam are naturally transmitted through the Asian citrus psyllid, \u003cem\u003eDiaphorina citri\u003c/em\u003e Kuwayama (Sternorrhyncha: Liviidae), while CLaf is transmitted by the African citrus psyllid \u003cem\u003eTrioza erytreae\u003c/em\u003e Del Guercio (Sternorrhyncha: Triozidae). HLB-infected trees show different symptoms including blotchy mottle and corky vein in leaves, defoliation, yellowing and die back as well as small misshapen fruits, with color inversion (Bove 2006).\u003c/p\u003e \u003cp\u003eThe Asian form of HLB disease has been observed in citrus growing areas in southern Iran in Sistan-Baluchistan, Kerman, Hormozgan, and Fars provinces and the presence of CLas in HLB-affected citrus trees has been confirmed employing DNA-based methods (Alizadeh et al. 2010; Faghihi et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Mohkami et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Salehi et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Salehi and Rasoulpour \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The disease is one of the causal agents of citrus decline in some areas of southern Iran, such as mandarin decline in Siyahoo in Hormozgan province of Iran (Faghihi \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDifferent strategies have been suggested in many citrus-producing countries with various results that have been indicated to reduce the development of HLB and help maintain tree productivity under field conditions. These measures include the use of insecticides and biological control to control psyllid vectors, improved foliar nutritional programs, the application of inorganic phosphorus (P) solution to the leaves, and the use of antibiotics like ampicillin, oxytetracycline, and streptomycin. Additionally, soil conditioners, plant defense inducers or activators, different graft/rootstock combinations, and thermotherapy may decrease the effects of HLB (Bassanezi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe development of citrus cultivars resistant to HLB is the most effective long-term control strategy for this damaging disease. Various research evaluated the effect of rootstock on the efficiency of citrus trees in the presence of HLB. Studies showed that the disease was transmitted to \u0026lsquo;Mosambi\u0026rsquo; budded on Lisbon lemon, Bengal citron, Carrizo citrange, Troyer citrange, and trifoliate orange, but HLB symptoms stayed mild in these combinations. Hence, application of tolerant rootstocks was suggested as a part of an integrated control program (Bassanezi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Hajivand et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) stated that no HLB symptoms were observed in the mandarin scions when grafted on \u003cem\u003eCitrus grandis\u003c/em\u003e rootstock with \u003cem\u003eCitrus hystrix\u003c/em\u003e as an interstock. The high vigor-inducing rootstocks, particularly Volker lemon, may enable younger infected trees to survive longer than others (Albrecht et al. 2012). Citrus cultivars and hybrids have variable reactions to HLB disease.\u003c/p\u003e \u003cp\u003eIn general, orange (\u003cem\u003eCitrus sinensis\u003c/em\u003e), tangerine (\u003cem\u003eC. reticulata\u003c/em\u003e) and their hybrids are very sensitive, grapefruit (\u003cem\u003eC. paradisi\u003c/em\u003e) and sour orange (\u003cem\u003eC. aurantium\u003c/em\u003e) are semi-sensitive, and \u003cem\u003ePoncirus trifoliata\u003c/em\u003e, Persian lime (\u003cem\u003eCitrus latifolia\u003c/em\u003e), Eureka lemon (\u003cem\u003eCitrus limon\u003c/em\u003e) and Mexican lime are tolerant to semi-tolerant to HLB disease (Chika et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; da Graca 1991; 2008; Deng et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Folimonova et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Some citrus species, such as \u003cem\u003eCitrus indica\u003c/em\u003e and \u003cem\u003eCitrus macroptera\u003c/em\u003e, did not develop disease symptoms under high inbreeding pressure and may be resistant to the disease (Bhagabati \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1993\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn Taiwan, the pomelo (\u003cem\u003eCitrus grandis\u003c/em\u003e) cultivar was previously resistant to HLB, but finally became infected and showed HLB symptoms about 30 years after HLB first appeared (Huang and Chang \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1980\u003c/span\u003e). Moreover, kumquat (\u003cem\u003eFurtunella margarita\u003c/em\u003e) eventually became infected with HLB disease, exhibiting mottling and yellowing symptoms (Tsai et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe final control of HLB is practical by identifying or producing resistant and tolerant citrus cultivars. The aim of this study was to investigate the reaction of three-year-old seedlings of commercial citrus cultivars and rootstocks in Iran to the causal agent of the Asian form of HLB disease (\u003cem\u003eCa\u003c/em\u003e. L. asiaticus) through grafting infection.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material and graft inoculation\u003c/h2\u003e \u003cp\u003eExperiments were conducted from September 2020 to September 2022 in a greenhouse where temperatures did not exceed 32\u0026deg;C. A number of commercial varieties of citrus in the country include Valencia sweet orange (\u003cem\u003eCitrus sinensis\u003c/em\u003e), Common sweet orange (\u003cem\u003eC. sinensis\u003c/em\u003e), Washington navel sweet orange (\u003cem\u003eC. sinensis\u003c/em\u003e), Blood sweet orange (\u003cem\u003eC. sinensis\u003c/em\u003e), Siyahoo mandarin (\u003cem\u003eCitrus reticulata\u003c/em\u003e), Clementine mandarin (\u003cem\u003eC. reticulata\u003c/em\u003e), Cleopatra mandarin (\u003cem\u003eCitrus reshni\u003c/em\u003e), Mexican lime (\u003cem\u003eCitrus aurantifolia\u003c/em\u003e), Bakraei (\u003cem\u003eCitrus\u003c/em\u003e spp.), Rangpur lime (\u003cem\u003eCitrus limonia\u003c/em\u003e), Rough lemon (\u003cem\u003eCitrus jambhiri\u003c/em\u003e), Macrophylla (Alemow) (\u003cem\u003eCitrus macrophylla\u003c/em\u003e), Volkamer lemon (\u003cem\u003eCitrus volkameriana\u003c/em\u003e) and Persian lime (\u003cem\u003eCitrus latifolia\u003c/em\u003e) were prepared and six seedlings from each were kept under controlled conditions. Prior to graft-inoculation, all studied plants analyzed by polymerase chain reaction (PCR) with the primers A2/J5 for \u0026lsquo;\u003cem\u003eCa.\u003c/em\u003e Liberibacter asiaticus/africanus\u0026rsquo;. The sources of inoculum were symptomatic branches of 3-year-old Valencia sweet orange seedlings which had previously been infected by CLas through graft-inoculation. Initial source of the infected grafts were mandarin trees severely affected by the Asian form of the HLB disease in an orchard in Siyahoo region (Hormozgan province, Iran).\u003c/p\u003e \u003cp\u003eTo evaluate citrus reactions to CLas infection, bark plus wood (budstick) in similar sizes (about 4 cm \u0026times; 1 cm) were taken from symptomatic branches and used as sources of inoculum. Two budsticks were grafted onto the stem of each plant in two places on the middle part of the seedlings with 10 cm distance. The grafting tissues were firmly fixed by wrapping them with transparent plastic strips for a month. Furthermore, to maintain moisture and promote graft growth, the graft-inoculated part of the plants was kept in a plastic bag for about two weeks. Three seedlings for each genotype were grafted with healthy (HLB-free) scions as negative control. All plants were kept in an insect-free greenhouse at temperatures ranging from 32\u0026deg;C and 14\u0026deg;C (day and night, respectively) and around 12 to 14 hours of natural light (Lopes et al. 2008).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePCR detection of CLas and disease incubation period\u003c/h3\u003e\n\u003cp\u003eThe inoculated plants were inspected regularly for symptom expression of the disease and transmission success of CLas examined at two months intervals up to two years by conventional PCR and nested-PCR analysis of total DNA extracted from leaf samples. Based on the symptom appearance and the incubation period of the disease as well as PCR results, the response of each genotype was evaluated to HLB disease.\u003c/p\u003e \u003cp\u003eThe cultivars were ranked from the most sensitive to the most tolerant, based on the average number of days between the time of grafting and the date of the first detection of CLas in the grafted seedlings of each cultivar. A completely random design (CRD) with three replications (for each genotype) was used and data analysis was done by analysis of variance (ANOVA).\u003c/p\u003e\n\u003ch3\u003eDisease severity measurement\u003c/h3\u003e\n\u003cp\u003e Disease severity was evaluated based on symptoms of HLB on the graft-inoculated seedlings according to the rating scale proposed by Hajivand et al (2010) with brief modifications. Based on the leaf symptoms, the scale included: 0\u0026thinsp;=\u0026thinsp;No symptom (Resistance), 1\u0026thinsp;=\u0026thinsp;Mild (mottling and interveinal chlorosis symptoms), 2\u0026thinsp;=\u0026thinsp;Moderate (Yellowing and/or Fe and Zn deficiencies like symptoms), 3\u0026thinsp;=\u0026thinsp;Severe (defoliation and twigs dieback symptoms)\u003c/p\u003e \u003cp\u003eDisease severity\u0026thinsp;=\u0026thinsp;Σ (a \u0026times; b) / N \u0026times; Z\u003c/p\u003e \u003cp\u003ewhere Σ (a \u0026times; b)\u0026thinsp;=\u0026thinsp;sum of the symptomatic plant and their corresponding rating, N\u0026thinsp;=\u0026thinsp;total number of sampled plants, and Z\u0026thinsp;=\u0026thinsp;highest rating.\u003c/p\u003e\n\u003ch3\u003eDNA Extraction and PCR assay\u003c/h3\u003e\n\u003cp\u003eDNA extraction was carried out according to the method of Hung et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1999\u003c/span\u003e. In order to detection of CLas in the plant samples, the conventional PCR using the specific primers OI1/OI2c (Jagoueixet al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) and a Nested-PCR using OI1/OI2c primer pair (ca. 1160 bp band) in the first round and CGO5R/CGO3F (Zhou et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) primer pair (ca. 800 bp band) in the second round were performed (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For both primer pairs, PCR was conducted in 35 cycles of 30 s at 94 \u0026deg;C (4 min at 95 \u0026deg;C for the initial denaturation), 40 s at 55 \u0026deg;C (60 \u0026deg;C for A2/J5 primer pair), and 80 s (1 min for A2/J5 primer pair) at 72 \u0026deg;C, and a final extension cycle of 10 min at 72 \u0026deg;C. The PCR reaction mixtures contained 10 \u0026micro;l of PCR Master Mix (Amplicon, Denmark), 0.4 pmol of each primer, and 2 \u0026micro;l of nucleic acid preparations. One microliter of diluted (1:10) PCR products from the first amplification was used as the template in the second round of PCR. DNA from healthy plants and sterile distilled water were used as the negative controls.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of primers used in conventional and nested PCR tests to detect \u003cem\u003eCa.\u003c/em\u003e Liberibacter asiaticus\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimer name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAnnealing (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAmplicon size (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTarget\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOI1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-GCGCGTATGCAATACGAGCGGCA-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e1160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e16S rDNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eJagoueix et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1996\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOI2c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-GCCTCGCGACTTCGCAACCCAT-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCGO3F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;- RGGGAAAGATTTTATTGGAG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e800\u003c/p\u003e \u003cp\u003e(nested-PCR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e16S rRNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eZhou et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2007\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCGO5R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-GAAAATAYCATCTCTGATATCGT-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;- TATAAAGGTTGACCTTTCGAGTTT-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e669 (CLaf)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eβ-operon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHocquellet et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1999\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJ5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;- ACAAAAGCAGAAATAGCACGAACAA-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e703 (CLas)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eElectrophoresis of both conventional PCR and Nested PCR amplified fragments was performed using 8 \u0026micro;l of amplified fragments loaded on 1% agarose gel containing 2% safe stain (Sinnaclon, Iran). Afterward, the banding patterns of amplified fragments were observed using UV transilluminator. Then, based on the presence or absence of the expected band, positive and negative reactions were recorded in each experiment.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eSymptomatology and incubation period of the disease\u003c/h2\u003e\n \u003cp\u003eThe inoculated plants were monitored in the greenhouse for two years, and different spectrums of disease symptoms were observed at various times post inoculation. These symptoms included mottling in the leaves, yellowing between the veins, symptoms similar to iron and zinc deficiency, yellow vein, and general yellowing of the plant (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eTable \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e separately shows the symptoms in different hosts at intervals of three months. According to the Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, no symptoms were observed in any of the commercial species and rootstocks of citrus until six months post inoculation (6 mpi). Eight and 9 mpi, disease symptoms appeared in one and two Valencia orange seedlings, respectively, while no symptoms were observed in other inoculated species like the control plants. About 12 mpi, the symptoms of the disease including mild mottling and yellowing between the veins in the upper leaves of the plant were observed in different cultivars of oranges and mandarins, Bakraei, Rangpur lime, and Alemow. However, no symptoms were observed in the seedlings of Mexican lime, Rough lemon, Volkamer lemon, and Persian lime 12 mpi. The symptoms of mild mottling and yellowing between the veins was observed during 16 to 19 mpi in Mexican lime, Rough lemon, Volkamer lemon, and Persian lime seedlings.\u003c/p\u003e\n \u003cp\u003eTable \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e shows the incubation period of the disease after graft-inoculation in different citrus species. The incubation period of HLB disease was varied among the citrus genotypes.\u003c/p\u003e\n \u003cp\u003eThe comparison of the average incubation period between genotypes showed that the highest incubation periods were observed in Mexican lime, Volkamer lemon, Rough lemon, and Persian limes, which had a significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with other citrus genotypes. No significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was observed in incubation period of the disease between Local orange, Washington navel oranges, Blood oranges, Local mandarin, Clementine mandarin, Cleopatra mandarins, Bakraei, Rangpur lime, and Alemow (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\u003cp\u003e\u0026lowast; These symptoms were observed in at least one of the three replicates inoculated for each genotype.\u003c/p\u003e \u003cp\u003eNo symptom (NS); Mild mottling (MM); Mottling (M); Interveinal chlorosis (IC); Fe and Zn deficiencies like symptoms (Fe/Zn Df); Yellowing in a part of the plants (PY); General yellowing (Y); Defoliation and die back (DD).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnalysis of variance related to the effect of citrus genotypes on incubation period of HLB disease.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSource of variation\u003c/p\u003e \u003cp\u003eS.V\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDegrees of freedom\u003c/p\u003e \u003cp\u003eD.F\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSums of squares\u003c/p\u003e \u003cp\u003eS.S\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean of Square\u003c/p\u003e \u003cp\u003eM.S\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF statistic\u003c/p\u003e \u003cp\u003eF.S\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCoefficient of variation\u003c/p\u003e \u003cp\u003eC.V%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e342.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30.7\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e7.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eError\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e366.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e**\u003c/sup\u003eSignificant at 1% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eMean comparison of the incubation period of HLB disease in citrus genotypes.\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCitrus genotypes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncubation period (month)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eValencia sweet orange\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.7\u003csup\u003e\u003cb\u003ec*\u003c/b\u003e\u003c/sup\u003e \u0026plusmn; 0.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCommon sweet orange\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.7\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWashington navel sweet orange\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.3\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlood sweet orange\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.3\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSiyahoo mandarin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.3\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClementine mandarin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.0\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCleopatra mandarin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.7\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMexican lime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.0\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBakraei\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.3\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRangpur lime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.7\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRough lemon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.7\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlemow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.7\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVolkamer lemon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17.3\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePersian lime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17.3\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLSD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.548\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e*\u003c/sup\u003e Means with at least one similar letter had no significant difference based on LSD at 5% of probability level.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePCR detection of\u003c/b\u003e \u003cb\u003eCa\u003c/b\u003e. \u003cb\u003eL. asiaticus in the genotypes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows the results of direct and nested-PCR tests in detection of \u003cem\u003eCa\u003c/em\u003e. L. asiaticus in the graft-inoculated plants at 60-day intervals. As can be seen, the sensitivity of the nested-PCR test in detecting CLas in the seedlings is higher than direct-PCR and could detect the infected plants at an earlier time. Statistical analysis and comparison of the average number of days between the time of graft-inoculation and the first time of positive detection of CLas by PCR in the seedlings showed that this period of time was significantly different between some citrus genotypes (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The average number of days until the first positive detection of CLas by PCR in citrus species showed that CLas was firstly detected in Valencia orange, i.e., 180 dpi (mean of direct and nested-PCR), and there was a significant difference (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between Valencia orange with several species, including Local orange, Blood orange, Cleopatra mandarins, Rough lemons, Rangpur limes, Volkamer lemons, and Persian lime (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In the studied species, the disease agent was lastly detected in Mexican lime and Persian lime (on average 330 dpi), and Volkamer lemon (on average 300 dpi). Nevertheless, there was no significant difference (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between Mexican lime, Persian limes, and Volkamer lemon in the number of days after inoculation until a positive detection in PCR (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Based on the average time between graft-inoculation and positive PCR detection of CLas in the inoculated species, it seems that Mexican lime, Persian lime, and Volkamer lemon had the most suppression against reproduction of CLas, because it was detected in these genotypes at a later time. Moreover, these genotypes had the highest incubation period of HLB as well as developed less severe HLB symptoms. Therefore, these genotypes are considered to be less susceptible to HLB than other citrus seedling verities. Rough lemon and Local orange were at the next level, that is, had the medium resistance to CLas. Alemow, Bakraei, Rangpur lime, Cleopatra mandarin, Clementine mandarin, Local mandarin, Blood oranges, and Washington navel orange were semi-sensitive, and Valencia orange was the most susceptible to CLas.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnalysis of variance of the individual and interaction effects of PCR method and citrus genotype on detection of \u003cem\u003eCa\u003c/em\u003e. Liberibacter asiaticus\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSource of variation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDegrees of freedom\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSums of squares\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean of Square\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF statistic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCoefficient of variation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCR method\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e75.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e75.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e60.8**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e158.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.79**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCR method \u0026times; Genotype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.6ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperimental Error\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e313.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e** and ns: Significant and non-significant at 1%, respectively\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean comparison of the number of days between grafting and HLB diagnosis times in different citrus cultivars\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eFirst positive PCR detection of CLas (day post inoculation)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDirect-PCR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNested-PCR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean (Host)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eValencia sweet orang\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003csup\u003e\u0026dagger;\u0026dagger;\u003c/sup\u003e220\u003csup\u003eefg\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e140\u003csup\u003eh\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003csup\u003e\u0026dagger;\u003c/sup\u003e180\u003csup\u003eE\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLocal sweet orange\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e300\u003csup\u003eabc\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e220\u003csup\u003eefg\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e260\u003csup\u003eBC\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWashington navel sweet orange\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e240\u003csup\u003edef\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e200\u003csup\u003efg\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e220\u003csup\u003eCDE\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlood sweet orange\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e280\u003csup\u003ebcd\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e200\u003csup\u003efg\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e240\u003csup\u003eCD\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLocal mandarin (Siyahoo)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e260\u003csup\u003ecde\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e180\u003csup\u003egh\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e220\u003csup\u003eCDE\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClementine mandarin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e260\u003csup\u003ecde\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e200\u003csup\u003efg\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e230\u003csup\u003eCD\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCleopatra mandarin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e280\u003csup\u003ebcd\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e200\u003csup\u003efg\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e240\u003csup\u003eCD\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMexican lime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e340\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e320\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e330\u003csup\u003eA\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBakraei\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e240\u003csup\u003edef\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e180\u003csup\u003egh\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e210\u003csup\u003eDE\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRangpur lime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e280\u003csup\u003ebcd\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e200\u003csup\u003efg\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e240\u003csup\u003eCD\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRough lemon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e320\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e260\u003csup\u003ecde\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e290\u003csup\u003eAB\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlemow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e260\u003csup\u003ecde\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e200\u003csup\u003efg\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e230\u003csup\u003eCD\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVolkamer lemon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e320\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e280\u003csup\u003ebcd\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e300\u003csup\u003eAB\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePersian lime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e340\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e320\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e330\u003csup\u003eA\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePCR method mean\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003csup\u003e\u003cb\u003e\u0026dagger;\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e281\u003c/b\u003e\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u003cb\u003e\u0026plusmn;\u0026thinsp;8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e221\u003c/b\u003e\u003csup\u003e\u003cb\u003eB\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u003cb\u003e\u0026plusmn;\u0026thinsp;9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eA= Each number is the average of 3 numbers related to 3 grafted seedlings of each cultivar. \u003csup\u003e\u0026dagger;\u003c/sup\u003eMeans in each left column and below row having capital (individual effect of each factor) and \u003csup\u003e\u0026dagger;\u0026dagger;\u003c/sup\u003ethe means in two middle columns having small letter (interaction of factors) with similar letters are not significantly different according to LSD test at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnalysis of variance in relation to Disease severity progress of HLB in different citrus cultivars\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS.V\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD.F\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS.S\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM.S\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF.S\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eProb.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.064\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e280.9**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCultivar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.054\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.081\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.04**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime \u0026times; Cultivar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.242\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.26ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eError\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.617\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.041\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ens, *,**\u003c/sup\u003e Not Significant, Significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDisease severity progress of HLB in different citrus cultivars\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eDisease severity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMean of Genotype\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12 months\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18 months\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24 months\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eValencia sweet orange\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003csup\u003e\u003cb\u003eΨ\u003c/b\u003e\u003c/sup\u003e0.3333\u003csup\u003e\u003cb\u003ede\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5555\u003csup\u003e\u003cb\u003ebc\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.7777\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003csup\u003e\u003cb\u003e\u0026dagger;\u003c/b\u003e\u003c/sup\u003e0.5555\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0668\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLocal sweet orange\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.2222\u003csup\u003e\u003cb\u003eef\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.4444\u003csup\u003e\u003cb\u003ecd\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5555\u003csup\u003e\u003cb\u003ebc\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.4074\u003csup\u003e\u003cb\u003eBC\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0586\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWashington navel sweet orange\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.2222\u003csup\u003e\u003cb\u003eef\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.4444\u003csup\u003e\u003cb\u003ecd\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5555\u003csup\u003e\u003cb\u003ebc\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.4074\u003csup\u003e\u003cb\u003eBC\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0586\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlood sweet orange\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.2222\u003csup\u003e\u003cb\u003eef\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.4444\u003csup\u003e\u003cb\u003ecd\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.6666\u003csup\u003e\u003cb\u003eab\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.4074\u003csup\u003e\u003cb\u003eBC\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0586\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLocal mandarin (Siyahoo)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.3333\u003csup\u003e\u003cb\u003ede\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5555\u003csup\u003e\u003cb\u003ebc\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.7777\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.5555\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0668\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClementine mandarin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.2222\u003csup\u003e\u003cb\u003eef\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.4444\u003csup\u003e\u003cb\u003ecd\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.6666\u003csup\u003e\u003cb\u003eab\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.4444\u003csup\u003e\u003cb\u003eB\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0717\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCleopatra mandarin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1111\u003csup\u003e\u003cb\u003efg\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.4444\u003csup\u003e\u003cb\u003ecd\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.6666\u003csup\u003e\u003cb\u003eab\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.4074\u003csup\u003e\u003cb\u003eBC\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0828\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMexican lime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0000\u003csup\u003e\u003cb\u003eg\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.2222\u003csup\u003e\u003cb\u003eef\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.4444\u003csup\u003e\u003cb\u003ecd\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.2222\u003csup\u003e\u003cb\u003eF\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0668\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBakraei\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.2222\u003csup\u003e\u003cb\u003eef\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.4444\u003csup\u003e\u003cb\u003ecd\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5555\u003csup\u003e\u003cb\u003ebc\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.4074\u003csup\u003e\u003cb\u003eBC\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0556\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRangpur lime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1111\u003csup\u003e\u003cb\u003efg\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.4444\u003csup\u003e\u003cb\u003ecd\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5555\u003csup\u003e\u003cb\u003ebc\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.3703\u003csup\u003e\u003cb\u003eBCD\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0717\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRough lemon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0000\u003csup\u003e\u003cb\u003eg\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.4444\u003csup\u003e\u003cb\u003ecd\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5555\u003csup\u003e\u003cb\u003ebc\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.3333\u003csup\u003e\u003cb\u003eCDE\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0888\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlemow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1111\u003csup\u003e\u003cb\u003efg\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.4444\u003csup\u003e\u003cb\u003ecd\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.6666\u003csup\u003e\u003cb\u003eab\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.4074\u003csup\u003e\u003cb\u003eBC\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0828\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVolkamer lemon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0000\u003csup\u003e\u003cb\u003eg\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.4444\u003csup\u003e\u003cb\u003ecd\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.4444\u003csup\u003e\u003cb\u003ecd\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.2963\u003csup\u003e\u003cb\u003eDEF\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0764\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePersian lime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0000\u003csup\u003e\u003cb\u003eg\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.3333\u003csup\u003e\u003cb\u003ede\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.4444\u003csup\u003e\u003cb\u003ecd\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.2592\u003csup\u003e\u003cb\u003eEF\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0693\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMean of Time\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003csup\u003e\u003cb\u003e\u0026dagger;\u003c/b\u003e\u003c/sup\u003e0.1508\u003csup\u003e\u003cb\u003eC\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.4365\u003csup\u003e\u003cb\u003eB\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0179\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5872\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0198\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cb\u003e\u0026dagger;\u003c/b\u003e \u003c/sup\u003eMeans having capital in the right column and below row with same letters are not significantly difference according to LSD test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cb\u003eΨ\u003c/b\u003e \u003c/sup\u003e Means having small letter in middle columns with same letters are not significantly difference according to LSD test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDisease severity\u003c/h3\u003e\n\u003cp\u003eBased on the disease symptoms, the severity of HLB disease was evaluated in different citrus hosts at 12, 18 and 24 months after CLas graft-inoculation (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). As shown in Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, 12 months post inoculation, HLB symptoms were not observed on Mexican lime, Rough lemon, Volkamer lemon and Persian lime, but mild HLB symptoms were observed on other hosts. The disease severity in the hosts increased over time and the most disease severity for each genotype was observed at 24 months post inoculation. After 24 months of inoculation, the lowest disease severity (0.44, moderate symptoms) was observed in Mexican lime, Volkamer lemon and Persian lime, and the highest disease severity (0.77, severe symptoms) was observed in Valencia orange and Local mandarin (Siyahoo).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe Asian form of citrus HLB caused by \u003cem\u003eCandidatus\u003c/em\u003e Liberibacter asiaticus was first found in Iran in 2008 (Faghihi et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The disease is transmitted by the Asian citrus psyllid, \u003cem\u003eDiaphorina citri\u003c/em\u003e, which is widespread in citrus growing areas in southern Iran (Alizadeh et al. 2022; Salehi et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this research, the reaction of three-year-old seedlings of 14 commercial cultivars and rootstocks of citrus to the causal agent of the Asian form of HLB disease (\u003cem\u003eCa\u003c/em\u003e. L. asiaticus) was investigated through graft-inoculation. Based on the PCR detection of CLas after grafting, the disease incubation period (the time between graft-inoculation and the appearance of the first symptoms of the disease), and disease severity in the seedlings, their reaction to the HLB disease were evaluated.\u003c/p\u003e \u003cp\u003eIn pathogen transmission through grafting, the primary inoculum present in the tissue of the scion is directly transferred to the tissue of the host plant. In this method, unlike the transmission with psyllid and \u003cem\u003eCuscuta\u003c/em\u003e spp., the exact time of inoculation, the amount of initial inoculum (the number and size of the scions), and the inoculation location can be controlled to a large extent (Batool et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). However, there are limitations in this transmission method, such as the survival of the scion, and the degree of compatibility of the scion tissue with the rootstock tissue.\u003c/p\u003e \u003cp\u003eThe symptoms observed in the infected citrus seedlings compared with the negative PCR seedlings (negative control) and the type of the symptoms in the inoculated plants was regularly recorded. Besides that, the PCR results confirmed the CLas infection in the seedlings. The symptoms of HLB disease are not very specific and the type and severity of the symptoms occurred in the greenhouse may be different from the symptoms in nature (due to the age of the trees, climatic conditions, and the presence of the vector). In nature, typical symptoms of the disease include yellowing shoots, blotchy mottle of leaves, corky veins, and localized symptoms of HLB spreads over the canopy, finally leading to defoliation and dieback. Moreover, the infected plants usually produce small, asymmetrical, bitter-tasting fruits with color inversion (Bove 2006). However, in our greenhouse experiment, only some of the symptoms were observed, and in many cases, the predominant symptoms in the infected seedlings were mottling, general yellowing, interveinal yellowing, and symptoms resembling iron and zinc deficiencies. The chlorotic pattern's symptoms similar to those of zinc and iron deficiencies can be seen in other diseases such as citrus tristeza, citrus stubborn, and phytoplasma infection. In the studied citrus genotypes, the symptoms of the disease appeared between eight and 18 months on different seedlings, and the incubation period varied between seedlings up to 10 months. On the other hands, the CLas could be detected by PCR in the inoculated plants several months before the appearance of the disease symptoms (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). For example, the first disease symptoms were noticed on Valencia orange (the most susceptible cultivar) at 8 to 9 mpi, while the first positive detection of CLas was found in Valencia orange seedlings by the nested-PCR method at 4 to 6 mpi and by the direct-PCR assay at 6 to 8 mpi. Therefore, on average, 2 to 4 months before the appearance of HLB symptom, the CLas was detected in inoculated Valencia orange seedlings by direct- and nested-PCR tests, respectively. Disease symptoms were appeared in other genotypes, such as another cultivar of oranges and tangerines, Bakraei, Rangpur lime, and Alemow, between 10 and 13 mpi. In these genotypes, the first positive PCR detection of CLas occurred between 6 and 8 mpi, or approximately 4 to 5 months prior to the onset of symptoms. The longest incubation period of the HLB disease was recorded between 16 and 19 mpi in the seedlings of Mexican lime, Rough lemon, Volkamer lemon, and Persian lime, while PCR confirmed that these genotypes were infected with CLas between 10 and 12 mpi. Hence, even in more tolerant plants, the CLas could be detected by PCR up to 12 mpi and several months before the appearance of the disease symptoms. It is difficult to detect CLas prior to the symptom expression, especially under natural conditions, due to its non-uniform distribution and low concentration in infected citrus trees. The highly sensitive nested-PCR is an appropriate approach for detecting CLas during the disease's incubation period and can reveal the presence of HLB infection several months before direct-PCR.\u003c/p\u003e \u003cp\u003eMolecular methods such as PCR have been used successfully to identify and detect the different species of \u003cem\u003eCa\u003c/em\u003e. Liberibacter (Jagoueixet al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Hocquellet et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). In some cases, direct-PCR may fail to detect the CLas in infected trees due to the low concentration of the bacterium or present of disturbing metabolites in the tissue extract. In this study, in addition to the direct-PCR, a sensitive nested-PCR was performed to detect the CLas. Overall, there was a significant difference between the nested-PCR and direct-PCR methods for detecting CLas in the inoculated plants. As expected, the nested-PCR had the higher sensitivity, and CLas was detected in the inoculated plants at least two months before direct-PCR. However, the false positive results may occur, especially in nested-PCR assay, hence DNA from a healthy plant and sterile distilled water should be used in each reaction as negative controls.\u003c/p\u003e \u003cp\u003eThe development of disease symptoms and the rate of CLas reproduction in host plant tissue reveal the different levels of sensitivity and tolerance of the host plant. Therefore, the faster the disease agent (CLas) can settle in the host and multiply, the sooner the pathogen is detected by the PCR method in the inoculated plants, and the earlier the disease symptoms appear, indicating that the host plant is more susceptible to the disease.\u003c/p\u003e \u003cp\u003eThe detection time of the bacterium in the phloem of the host plant, along with the appearance time of the disease symptoms, could be an appropriate relative index to evaluate the response of different citrus cultivars to the pathogen and compare them with each other. Thus, an earlier detection of CLas in some hosts, such as Valencia orange, indicates better reproduction and a faster increase in the concentration of the bacterium until reaching the detecting threshold by PCR. Furthermore, a correlation was found between the time of the positive PCR detection of CLas after graft inoculation, disease incubation period, and disease severity in the examined cultivars and rootstocks. It means that the more sensitive inoculated seedlings became rapidly positive in PCR due to the higher concentration of the pathogen, and the symptoms of the disease appeared earlier (shorter incubation period) and disease severity was higher. The plant hosts with long disease incubation period and mild symptoms were considered less susceptible to HLB disease.\u003c/p\u003e \u003cp\u003e According to the investigations, the concentration of the pathogenic bacterium in the plant is not uniform, and it is higher in the symptomatic parts of the infected plant. This demonstrates the enhanced proliferation of the bacterium in the symptomatic parts of the plant (Kunta and de Graca 2014).\u003c/p\u003e \u003cp\u003eBased on the time of the positive PCR detection of CLas and the symptom appearance of the disease, Mexican lime, Persian lime, and Volkamer lemon placed at the highest tolerance level (lowest susceptibility), and Rough lemon and Local orange were at the next level, i.e., semi-tolerance (medium susceptibility). The next group included Alemow, Bakraei, Rangpur lime, Cleopatra mandarin, Clementine mandarin, Local mandarin, Blood orange, and Washington navel orange, which were classified as susceptible to the disease, and the last group included the Valencia orange, which was the most susceptible to the disease.\u003c/p\u003e \u003cp\u003eIn the surveys in the citrus growing areas in southern Iran, it has been observed that the Asian citrus psyllid (\u003cem\u003eDiaphorina citri\u003c/em\u003e) has a host preference for Mexican lime and establishes a larger population on it, but the lower severity of the HLB disease has been found on Mexican lime trees. Under natural conditions, the incidence and severity of HLB are considerably higher in orange and mandarin trees than in other citrus species, despite the fact that \u003cem\u003eD. citri\u003c/em\u003e populations on these trees are much smaller than those on Mexican lime.\u003c/p\u003e \u003cp\u003eSeveral studies have shown that orange and tangerine cultivars are sensitive, grapefruits and sour orange are semi-sensitive, and lemons are more tolerant to the HLB disease (Folimonova et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Halbert and Manjunath \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Miyakawa and Zhao \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Wild genotypes of citrus, such as \u003cem\u003eCitrus indica\u003c/em\u003e and \u003cem\u003eCitrus macroptera\u003c/em\u003e in Northeast India, remained asymptomatic despite infected psyllid in the region (Bhagabati \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Poncirus trees and seedlings (\u003cem\u003ePoncirus trifoliata\u003c/em\u003e) are also very resistant to this disease (Miyakawa \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1980\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMexican lime had relatively high tolerance against the disease, and uses as a rootstock in different parts of Iran. Persian lime is tolerant to both HLB and lime witches\u0026rsquo; broom, making it suitable for planting in locations such as southern Iran where both diseases prevail.\u003c/p\u003e \u003cp\u003eThere have also been reports of the relative resistance of several rootstocks to this disease.\u003c/p\u003e \u003cp\u003eIn conclusion, all the studied citrus genotypes, irrespective of their rootstock, were susceptible to the HLB disease. However, symptoms vary from cultivar to cultivar, with the most severe found on Valencia sweet orange, and Local mandarin. Symptoms were less severe on Mexican lime, Persian lime and Volkamer lemon.\u003c/p\u003e \u003cp\u003eThe studies suggest that proper tolerance to HLB, with reduced symptoms and probably reduced CLas titer might be usually found in rootstock cultivars and symptomless natural biotypes. These sources can be used as rootstocks, and for breeders to produce resistant cultivars. Due to the lack of natural resistance in citrus species, employing genetic engineering to create HLB-resistant commercial cultivars would have a high chance of success (Bove 2006).\u003c/p\u003e \u003cp\u003eOver the last few years, research has focused on genome-editing technologies such as CRISPR/Cas9 and single-guide RNA (sgRNA) to enhance crop development. CRISPR/Cas9 is a specific and straightforward technique used to modify the genome of crops to produce disease-resistant/tolerant plants, and it might be the most accepted approach for crop improvement in the future (Ghosh et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurther work is needed to assess investigating the response of more citrus genotypes to CLas. Furthermore, in the citrus genotypes, CLas concentration and multiplication process can be checked in different organs especially root system and compared with each other using quantitative real time-PCR.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor Contribution\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShiva Safarpour Kapourchali\u003c/strong\u003e: Writing \u0026ndash; original draft, Methodology, Investigation. \u003cstrong\u003eMohammad Mehdi Faghihi\u003cstrong\u003e:\u003c/strong\u003e\u003c/strong\u003e Writing \u0026ndash; review \u0026amp; editing, Conceptualization, Methodology, Investigation, Formal analysis. \u003cstrong\u003eNazanin Sadat Ebadi:\u003c/strong\u003e Methodology, Investigation \u003cstrong\u003eAli Alizadeh Aliabadi\u003cstrong\u003e:\u003c/strong\u003e\u003c/strong\u003e Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eHamed Hasanzadeh Khankahdani:\u0026nbsp;\u003c/strong\u003eSoftware, Methodology, Formal analysis. \u0026nbsp; All the authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Islamic Azad University of Iran, Varamin-Pishva Branch, and Fars Agricultural and Natural Resources Research and Education Center for supporting this work.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlbrecht U, Hall DG, Bowman KD (2014) Transmission efficiency of \u0026lsquo;\u003cem\u003eCandidatus\u003c/em\u003e Liberibacter asiaticus\u0026rsquo; and progression of huanglongbing disease in graft and psyllid-inoculated citrus. Hort Sci 49:367\u0026ndash;377\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlizadeh Aliabadi A, Foroutan A, Golmohamadi M (2010) Occurrence of citrus greening caused by \u003cem\u003eCandidatus\u003c/em\u003e Liberibacter asiaticus in Sistan-Baluchestan province. Proc 19th Iranian Plant Protection Congress, 31 July\u0026ndash;3 August 2010. Tehran, Iran, p 525. (in Persian with English summary)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlizadeh Aliabadi A, Faghihi MM, Salehi M, Ghasemi A (2022) Dynamics of emergence and spread of citrus huanglongbing disease in Iran. Plant Pathol Sci 11(2):11\u0026ndash;21\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBassanezi RB, Lopes SA, de Miranda MP, Wulff NA, Volpe HXL, Ayres AJA (2020) Overview of citrus huanglongbing spread and management strategies in Brazil. Trop Plant Pathol 45:251\u0026ndash;264\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBatool A, Iftikhar Y, Mughal SM, Khan MM, Jaskani MJ, Abbas M, Khan IA (2007) Citrus greening disease \u0026ndash; a major cause of citrus decline in the world: a review. Hort Sci (Prague) 34:159\u0026ndash;166\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhagabati KN, Moreno P (1993) JV da Gra\u0026ccedil;a LW, Timmer (eds), University of California, Riverside, 441\u0026ndash;442\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoina DR, Bloomquist JR (2015) Chemical control of the Asian citrus psyllid and of huanglongbing disease in citrus. Pest Manag Sci 71:808\u0026ndash;823\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBov\u0026eacute; JM (2000) Genomic characterization of a Liberibacter present in an ornamental rutaceous tree, Calodendrum capense, in the Western Cape province of South Africa. 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In Molecular and Diagnostic Procedures in Mycoplasmology 2:369\u0026ndash;380. Edited by S. Razin \u0026amp; J. G. Tully. San Diego: Academic Press\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSechler A, Schuenzel EL, Cooke P, Donnua S, Thaveechai N, Postnikova E, Stone AL, Schneider WL, Damsteegt VD, Schaad NW (2009) Cultivation of \u0026lsquo;\u003cem\u003eCandidatus\u003c/em\u003e Liberibacter asiaticus\u0026rsquo;,\u0026lsquo;\u003cem\u003eCa.\u003c/em\u003e L. africanus\u0026rsquo;, and \u0026lsquo;\u003cem\u003eCa.\u003c/em\u003e L. americanus\u0026rsquo; associated with huanglongbing. Phytopathology 99:480\u0026ndash;486\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsai CH, Su HJ, Liao YC, Hung TH (2006) First report of the causal agent of huanglongbing (\u003cem\u003eCandidatus\u003c/em\u003e Liberibacter asiaticus) infecting kumquat in Taiwan. Plant Dis 90:1360\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVillechanoux S, Garnier M, Renaudin J, Bov\u0026egrave; JM (1993) The genome of the non-cultured bacterial-like organism associated with citrus greening disease contains the \u003cem\u003enusGrplKAJL-rpoBC\u003c/em\u003e gene cluster and the gene for a bacteriophage DNA polymerase. Curr Microbiol 26:161\u0026ndash;166\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou LJ, Gabriel DW, Duan YP, Halbert SE, Dixon WN (2007) First report of dodder transmission of huanglongbing from naturally infected \u003cem\u003eMurraya paniculata\u003c/em\u003e to citrus. Plant Dis 91:227\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table 2","content":"\u003cp\u003eTable 2 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Citrus cultivars, Disease severity, Huanglongbing, Susceptibility","lastPublishedDoi":"10.21203/rs.3.rs-8463450/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8463450/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCitrus huanglongbing (HLB) associated with \u003cem\u003eCandidatus\u003c/em\u003e Liberibacter spp. is the most destructive bacterial disease of citrus crops worldwide. In this study, the response of three-year-old seedlings of 14 commercial citrus cultivars and rootstocks to the Asian form of HLB disease (\u003cem\u003eCa\u003c/em\u003e. L. asiaticus) was investigated through graft-inoculation. The seedlings' reactions to the HLB disease were evaluated based on the PCR detection of CLas after grafting, the disease incubation period (time between graft-inoculation and appearance of the first disease symptoms), and disease severity. Depending on the time of the positive PCR detection of CLas and appearance of disease symptoms, Mexican lime, Persian lime and Volkamer lemon were ranked at the highest level of tolerance (lowest susceptibility), and Rough lemon and Local orange were at the next level, i.e., semi-tolerance (intermediate susceptibility). Alemow, Bakraei, Rangpur lime, Cleopatra, Clementine mandarin, Local mandarin, Blood orange, and Washington navel orange were classified as susceptible to the disease, followed by Valencia orange, which was the most susceptible. In conclusion, all citrus genotypes studied, were affected by HLB. However, symptoms varied by cultivar, with the most severe observed on Valencia sweet orange and Local mandarin, while less severe symptoms were observed on Mexican lime, Persian lime and Volkamer lemon.\u003c/p\u003e","manuscriptTitle":"Reaction of some commercial citrus genotypes to Candidatus Liberibacter asiaticus in Iran","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-24 15:16:02","doi":"10.21203/rs.3.rs-8463450/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0a3a3546-333a-42e2-a01f-526bdc7c5239","owner":[],"postedDate":"February 24th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Reject after review","date":"2026-05-07T13:44:18+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-07T17:44:56+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-24 15:16:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8463450","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8463450","identity":"rs-8463450","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}