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Certain compounds of plants such as essential oils, with insecticidal properties have been considered as alternatives to chemical pesticides for pest control. This study reports the fumigant toxicity of Citrus reticulata Blanco (Rutaceae) peel essential oils against stored-product insect pest, red flour beetle, Tribolium castaneum Herbst (Tenebrionidae) adults. Experiment was carried out at 27 ± 1 ^°C and 60 ± 5 % relative humidity in darkness. Experimental concentrations were 15, 22, 31, 45, and 63 μl/l air tested on adult (1-7 days old) insects after 24 and 48 h of exposure. Results indicated that essential oils from C. reticulata had fumigant tocxicity effects against this stored pest. LC50 values were 38.2 and 35.6 μl/l air at 24 and 48 h after exposure of T. castaneum adults respectively. The essential oils of Citrus reticulata fruit peels at the highest dose of 63 μl/l air caused 76.6 % and 79 % mortality of insects after 24 and 48 hours of exposure, respectively. Mortality of T. castaneum increased with both increase in concentration of C. reticulata oils as well as exposure time of treated insects. These results suggest the potencial of C. reticulata oil as a control agent against T. castaneum.  

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The citrus leafminer, Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae) is a major invasive pest of citrus in Tunisia. In order to help the implementation of an efficient integrated management strategy, it was essential to assess the genetic diversity and population structure of the pest. For this purpose, random-amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) was applied, using eight oligo-nucleotide primers, to reveal genetic variability among eight populations of P. citrella, originating from the north, center and south of Tunisia. A total of 66 RAPD markers and 33 phenotypes were generated. Inter-population polymorphism was revealed, using the percentage of polymorphic markers (62.12 %), mean number of phenotypes generated per primer (4.125) and mean genetic distance (0.199). Hierarchical analysis, using the UPGMA method, indicated that the genetic variability was influenced by the regional distribution. This pattern of population clustering was supported by Principal Coordinate Analysis (PCO). Yet, a weak correlation (0.69) was revealed between genetic and geographic distances, suggesting that climatic contrariety between the north and south of Tunisia plays a major role in the differentiation of P. citrella, leading to a restriction of gene flow between populations. Results obtained in this work show clear genetic differences, which should be considered in the development of control strategies.  

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Asiatic citrus canker is a devastating disease resulting in drastic economic losses in citriculture worldwide. Amongst three different types of the disease, i.e. A, A* and A^w, the A* type is genetically less known. In order to comprehend the behavior of the Asiatic citrus canker A*-type strain (Xanthomonas citri subsp. citri) in the vicinity of the host cells, a targeted semi-quantitative transcript analysis approach via RT-PCR was carried out. A subset of sixteen genes, as representative of different steps involved in phytopathogencity, was analyzed on the culture medium (as uninduced) and compared with the subset isolated from the infected Mexican lime (Citrus auarntifolia L.) plants (as induced). The results showed that certain genes were up-regulated in induced condition, suggesting a putative role in bacteria-host interaction. Furthermore, the transcripts in induced condition could be classified into constitutive, early- and late-responsive genes, demonstrating their functional relevance during the host-pathogen interaction.    

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Samples of leaf, twig and fruit from ‘Mexican’ lime (Citrus aurantifolia) and grapefruit (Citrus paradisi) with symptoms of bacterial canker were collected from different provinces throughout Iran during spring and summers of 2010 and 2011. Yellow, gram-negative colonies were isolated from infected tissue samples. Results of pathogenicity assays indicated that some isolates incited tissue hyperplasia, hypertrophy and raised callus-like lesions typical of canker in hosts while other isolates stimulated flat necrotic and water-soaked lesions on leaves. Candidate samples of each group were identified according to morphological and physiological characteristics. Detections were also made using specific primers and partial sequencing of 16SrDNA for Pantoea group and gyrB for Xanthomonas group. Results showed that one group was characterized as the typical Xanthomonas citri subsp. citri strain while the other group containing most of the isolates was identified as Pantoea agglomerans. Samplings done frequently in different seasons revealed the presence of high populations of P. agglomerans with bacterial canker, especially in warmer and drier regions. These bacteria were able to incite canker-like symptoms on grapefruit seedlings and could be reisolated after two months.

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A survey was carried out in citrus nurseries in Mazandaran and Kerman provinces as the major citrus growing regions of Iran. Different varieties of orange and tangerine with abnormal symptoms were examined for the occurrence of Candidatus Liberobacter asiaticus, Spiroplasma citri and phytoplasma as the most important vascular limited pathogens in citrus. By visual inspection and molecular examination of 360 samples for the presence of fastidious prokaryotes only an occasional occurrence of phytoplasma and Spiroplasma citri were detected, using phytoplasma universal primer pairs P1/P7 and fU5/rU3 (in single tube nested PCR assay) and Spiroplasma specific primers P89-r/f and P58-6f/4r. PCR products of phytoplasmas were digested with MseI, AluI, BamHI and TaqI restriction enzymes and the digestion results showed phytoplasma related to aster yellows phytoplasma group. No amplicon was obtained for Candidatus Liberobacter asiaticus. The findings suggest when pathogen’s titer is low; symptoms of vascular limited diseases are almost confused with disorder and deficiency signs. So for sanitary selection and certification program, use of sensitive detection methods is recommended. Multiplex PCR assay used in this study successfully detected Candidatus Liberibacter asiaticus, Spiroplasma citri and phytoplasma in one reaction and is suggested for health certification programs.  

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A faunistic survey was conducted to identify the leafhoppers in the southwest of Iran (Khouzestan Province) in 2014. Among the collected materials, Aconurella nuristana Dlabola, 1957 is recorded for the first time for Iranian leafhoppers fauna. Diagnostic characters and geographical distribution of the newly recorded species is presented briefly.

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The red flour beetle, Tribolium castaneum (Herbst), is one of the most destructive pests attacking stored grain products all over the world. Serious problems assossiated with using synthetic chemical insecticides have strongly demonstrated the need for applying alternative safe compounds such as plant essential oils. The present experiment was conducted to evaluate fumigant toxicity of essential oils from the fresh fruit peel of two Citrus species namely, Citrus sinensis (L.) Osbeck and Citrus paradisi (Macfarlane) against 1 to 7-days-old adults of T. castaneum under laboratoryconditions. Moreover, oviposition deterrence activity of sublethal concentrations of the oils were assessed on the female beetles. All experiments were carried out at 27 ± 1 °C and65 ± 5% r. h. in darkness. Findings indicated the high fumigant toxicity of both essential oils. According to probit analysis, there was no significant differences between C. sinensis (LC₅₀ = 7.27 µl.l^-1 air) and C. paradisi (LC₅₀ = 7.70 µl.l^-1 air) essentialoils. Also, oviposition deterrence activity of the essential oils was significantly increased as concentrations of the oils were increased from 500 to 2500 ppm. In general, the results of our study demonstrated the high efficacy of C. sinensis and C. paradisi oils against T. castaneum.
 

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Huanglongbing (HLB) also known as citrus greening, is a destructive disease of citrus and now, is considered as a new emergence and spread out threat to the Middle East and North Africa (MENA) citrus production. In a survey conducted in southern Iran in 2013-2014, 77 citrus samples exhibiting symptoms of HLB were collected. Single-step and nested polymerase chain reactions (PCR) were employed to determine the presence of the phloem-limited bacterial pathogens ‘Candidatus Liberibacter asiaticus’(CLas), phytoplasma and Spiroplasma citri. Both CLas and phytoplasma were detected in HLB-affected citrus trees as co-infection (7.79%) and single infection (10.38% for phytoplasma and 42.85% for CLas). According to the proposed 16S rDNA-based phytoplasma classification scheme, the HLB-associated phytoplasma from this study was a member of peanut witches’broom (16SrII) phytoplasma group. This is the first report of association of a phytoplasma with HLB in sweet lime in the world and first record of association of CLas with sour orange (Citrus aurantium L.) and sweet lime in Iran.

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Citrus swallow tail butterfly, Papilio demoleus L. is a pest of citrus in many parts of the world. We examined the effect of four different citrus hosts including Citrus sinensis (L.) (Tarocco and Valencia) Citrus limon (L.) (Eureka), and Citrus paradisi Macfad. (Shambarg) on its life table parameters and biological characteristics at 24 ± 1 °C, 75 ± 5% RH and a photoperiod of 16: 8 h (L: D). The developmental time of immature stages was longest onC. sinensis (Tarocco) (12.3 ± SE days).The intrinsic rate of natural increase was significantly affected by variouscitrus, being the highest on C. sinensis (Tarocco) (0.099 day^-1) and lowest on C. sinensis (Valencia) (0.011 day^-1).The highest and lowest finite rates of increase were observed onC. sinensis(Tarocco) 1.10 andC. sinensis(Valencia)1.01, respectively.The highest gross reproductive rate and the lowest mean generation time were observed on C. sinensis (Tarocco) and C. sinensis. (Valencia), respectively. The findings indicated that C. sinensis (Tarocco) appeared to be the most susceptible host to P. demoleus among the tested varieties. The results of this and similar studies in the future on other hosts may be considered as a basis for finding resistant host/hosts in monitoring this pest.

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In this study the biodiversity and relative abundance of Collembola was evaluated in two different ecosystems, citrus field and forest in Mazandaran province, Iran. Samples were carried out during 2016 and 2017. In each site, soil was sampled by a collecting core from 5×5 cm in surface area and a depth of 10 cm. Species richness, biodiversity and the indices of similarity were calculated. The results of T-Test showed that in two years (2016 and 2017), the values of Shannon-Wiener diversity and Simpson species diversity index were significantly different between sampling areas (Pvalue<0.05), but the values of Margalef richness and Pielou evenness index were not significantly (Pvalue˃0.05). In the year 2016, Shannon-Wiener index, Simpson index, Pielou evenness and Margalef species richness in Dasht-e Naz forest were 1.92 ± 0.1, 0.83 ± 0.03, 2.01 ± 0.2 and 0.94 ± 0.04 respectively, and in citrus field were 1.44 ± 0.11, 0.73 ± 0.02, 1.46 ± 0.37 and 0.9 ± 0.03 respectively. Also, in the year 2017, Shannon-Wiener index, Simpson index, Pielou evenness and Margalef species richness in Dasht-e Naz forest were 1.79 ± 0.05, 0.81 ± 0.08, 1.38 ± 0.25 and 0.94 ± 0.09 respectively, and in citrus field were 0.98 ± 0.22, 0.58 ± 0.08, 1.02 ± 0.35 and 0.95 ± 0.05 respectively. In general, the results showed that the abundance and biodiversity of Collembola in Dasht-e Naz forest was higher than in citrus field.

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The Xanthomonas citri pv. citri (Xcc) is causal agent of bacterial citrus canker which is major disease of citrus throughout the world. The pthA bacterial effector protein is presented within the infected plants and indispensable of canker. The scFv antibodies are valuable tools for diagnosis and suppression of pathogens within plants. The present article describes developing and characterization of specific recombinant monoclonal scFv antibodies against pthA effector protein. For this aim, the gene encoding pthA protein was heterologously expressed in Escherichia coli and used for screening of Tomlinson phage display antibody library to pinpoint specific single chain variable fragment (scFv). In each round of panning, the affinity of phage towards pthA was checked by enzyme linked immunosorbent assay (ELISA). The data was indicative of about 50% of the monoclonal phages to be reactive strongly against pthA protein. Among the positive clones, 5 samples (A12, B8, C1, H8 and G8) were capable of detecting Xcc-infected plant samples and recombinant pthA protein. Restriction fragment length polymorphism showed similar banding pattern for all 5 scFvs as renamed to pthA-scFG8. HB2151 E. coli cells were infected by the phage bearing pthA-scFG8, and the expression of the peptide was induced by IPTG to produce a 30 kDa recombinant molecule. I-TASSER was used for homology modeling of both scFv and pthA and docking was carried out by Hex program. The latter demonstrated binding energy of −784 kcal/mol in scFv-pthA. 

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Citrus disinfection has the major effect to prevent fungal attacks during storage. In this research, Thompson orange fruit was disinfected with ortho phenyl phenol solution by thermal fogging machine and then some of the samples coated by Carnuba wax. Physicochemical properties of orange samples (disinfected; disinfected-wax and control samples) were evaluated during three months storage. Factorial experiment design was selected to find the effect of coating and storage time on orange quality properties. The results showed that the effect of coating on all measured quality properties, and the effect of coating and storage time on pH values were significant (p<0.01). The amount of pH value of disinfected sample was significant (p<0.05) and was higher than the rest of the samples. So disinfection treatment enhanced fruit resistance against fungal attacks. Amount of total soluble solid and acid total of fruit juice for disinfected-wax samples decreased and increased during storage, respectively, which this trend could indicatefermentation phenomenon in the fruit. Therefore, disinfection with ortho phenyl phenol solution by thermal fogging machine was effective and preserved the fruit quality against fungal diseases during storage period.

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Hop stunt viroid (HSVd) isolates have been reported as the causal agent of citrus cachexia in Mazandaran Province and recently shown to be associated with yellow corky vein disease of sweet orange and split bark disorder of sweet lime in the Fars Province, Iran. In the present work isolation and partial characterization of viroids from citrus trees affected by gummy stem blight is reported from Kohgiluyeh–Boyerahmad (KB) Province of Iran. Fifteen samples of citrus trees from Dehdasht area (KB Province) showing bark necrosis, gum exudation and die-back as well as seven citrus symptompless trees from the same area were tested for the prevalence of viroids, through Reverse Transcription Polymerase Chain Reaction (RT-PCR) followed by sequencing of PCR products. They were also tested for Citrus tristeza virus through Double-Antibody Sandwich Enzyme Linked Immunosorbent Assay (DAS-ELISA). Two variants of HSVd which differed from GenBank isolates in nucleotide sequence and two variants of Citrus Bent Leaf Viroid (CBLVd) were identified in any of the symptomatic samples. Moreover, a Citrus Exocortis Viroid (CEVd) was found only in symptomatic sweet lime. An HSVd isolate from KB (HSVd-bn₁) was selected and used for comparison with a number of HSVd variants from Iran (Fars and Mazandaran Provinces) and the related accessions from GenBank. On the basis of nucleotide sequence and secondary structure analysis, HSVd-bn1 and HSVd-bn2 belong to non-cachexia variants of HSVd and have about 95% similarity to Citrus gummy bark viroid, a sub-species of HSVd. CTV was not detected in the diseased plants. It is yet to be determined whether bark necrosis of sweet lime and of sweet orange plants is caused solely by the associated viroid(s) or other factors are involved as well.

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The effects of rootstocks such as 'sour orange' (Citrus aurantium L. var. ‘Yerli’), 'Carrizo' and 'Troyer citranges' (Citrus sinensis Osb. x Poncirus trifoliata Raf), 'Smooth Flat Seville sour orange' (Citrus spp. hybrid of uncertain origin), 'Brazilian sour orange' (Citrus aurantiam L. var. ‘Brasilian’), 'Volkameriana' (Citrus volkameriana Tan. and Pasq.) and 'Calamondin' (possibly Citrus reticulate var. austere×Fortunella hybrid, Swingle) on plant growth, fruit yield, and quality of ‘Rio Red’ grapefruit were investigated from 2008 to 2012. Rootstocks were found to have significant effects on plant growth, fruit yield, and quality. ‘Rio Red’ grapefruit trees budded on 'Carrizo' and 'Troyer citranges' showed higher vegetative growth parameters (canopy height, diameter, and volume) than the trees on the other rootstocks. The trees on 'Volkameriana' and 'Carrizo citrange' produced higher percentage of cumulative yield of about 55.1 and 34.3%, respectively, than the trees on 'sour orange'. ‘Rio Red’ grapefruit budded on 'Carrizo citrange' had higher fruit quality such as thin rind, high juice content, and more color development than the other rootstocks. 'Carrizo citrange' was the most promising rootstock for ‘Rio Red’ grapefruit in Dörtyol, Eastern Mediterranean region of Turkey.

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To understand the relationship between natural physicochemical properties of soil and abundance of citrus root nematode (Tylenchulus semipenetrans), a survey was conducted during 2009-2010 in some 37 citrus orchards, in Kohgilouyeh va Boyerahmad Province, Iran. Distribution of the citrus nematode was determined by collecting random samples from the soil and citrus plant roots, extracting and enumerating the number of second stage juveniles, males/100 g of soil and females/5 g of root. The relationships between nematode population, and the factors of: organic matter content, Nitrogen (N), Phosphorus (P), Potassium (K), organic carbon, Calcium Carbonate, soil texture, Electrical Conductivity (EC) and pH were determined. Increase in nematode population density was observed by increasing soil saturation percentage (up to 43%), and by an increase in soil silt, sand, P, K and organic carbon, but by an increase in soil salinity, Calcium Carbonate, total Nitrogen and the amount of clay in soil, the nematode population decreased. Maximum nematode population density was recorded in a loamy soil texture. The most suitable soil pH for nematode activity was found almost seven while either an increase or decrease in soil pH, resulted in a decrease in nematode population. The number of second stage juveniles ranged from 58 to 2,730/100 g of soil while females were present by 11 to 331 individuals/g of root respectively. About 62% of the studied orchards were infested with T. semipenetrans.

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One of the really important challenges facing agricultural producers is the choice of their product distribution channels. The present study is aimed at investigating the citrus marketing channel strategy and its determinants among citrus orchardists of Mazandaran using a Nested Logit model. Some two hundred and fifty two orchardists from 15 cities of Mazandaran were taken as the study samples. Four distribution channels and three nests were considered in determining the factors influencing the choice of marketing channel. Since the estimated structural parameter did satisfy the condition of lying between 0 and 1, the nested logit model was proved as appropriate for the intended application. The results indicated that the orchard distance from the nearest city, orchardist’s experience, sale time, marketing costs, the kind of product and the dominant marketing channel constitute the most important factors of distribution channel selection as regards the citrus producers in Mazandaran. According to the obtained results, by increase in the orchards’ distance from the nearest city, the producers' tendency to sell their products through channels of shorter distances from buyers also increases. Also, results revealed that the older the orchardist, the higher his/her degree of risk aversion. An estimation of binomial Logit Model for the second and third nests had consistent results regarding the marketing costs. As the marketing costs increases, the orchardist’s tendency to sell his/her products through other channels of a lower marketing cost will increase. Furthermore, results revealed that as the citrus price rises, the producers get further intended to sell their products in the market rather than selecting either the pre-sale method or selling their produce to the local dealers.

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The decay of green and blue molds of Thomson navel oranges is one of the main causes of this product's decay and it can be a limiting factor in storing fruits. The aim of this study was to investigate the effects of wax, hot water, calcium chloride and commercial Tecto 60 fungicide treatments on reducing decay by inducing biosynthesis of flavonoid compounds and maintaining the postharvest quality of Thomson navel orange fruits. Fruits kept for three months in a cold storage at a temperature of 5 to 7 °C and a relative humidity of about 75 to 90%. Weight loss of fruits in waxed fruits was significantly lower than other treatments. The wounded and infected fruits did not have much chance of survival. None of the treatments were significantly effective on control of decay of non-wounded fruits. Total phenol content of fruit peel and flesh  decreased during storage. Hesperidin in the peel was at the highest level in Britex wax treatment. The highest amount of scoparon of fruit peel and flesh was in hot water and calcium + wounded treatments, respectively. Although the amount of scoparon in some treatments inhibited the germination of fungal spores, it did not have the expected effect in controlling decay. The use of Tecto 60™ and hot water can significantly reduce the decay of wounded and infected fruits. The treatments were not effective in controlling the deterioration of intact fruits, which indicates the importance of preventing mechanical damage to the fruit in the pre-harvest stage.

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In this study, the antimicrobial effect of Citrus aurantium essential oil on Bacillus cereus, Listeria innocua, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi and Candida albicans was evaluated. The chemical compounds, total phenol content, total flavonoids content and antioxidant potential of Citrus aurantium essential oil were determined. The results of phytochemical analysis (Ferric chloride and Shinoda) showed that, phenol, flavonoids and flavone demonstrated the presence in Citrus aurantium essential oil. Based on gas chromatography–mass spectrometry results 19 compounds identified in Citrus aurantium essential oil. Linalool (21.29 %) was the major compound in Citrus aurantium essential oil. The total phenolic content and total flavonoids content of Citrus aurantium essential oil were equal to 41.35 ± 0.46 mg GAE/g DM and 2.98 ±0.50 mg QE/g DM, respectively. The antioxidant potential based on radical Scavenging and β-carotene linoleic acid of Citrus aurantium essential oil were equal to 102.85 ± 0.60 μg/ml and 65.60% respectively. The longest and the shortest diameters of the inhibition zone at the concentration of 45 mg/ml pertained to, Listeria innocua (16.50 ±0.66 mm) and Salmonella typhi (9.80 ±0.43 mm), respectively. The minimum inhibitory concentration of the Citrus aurantium essential oil ranged from 3.125 mg/ml to 100 mg/ml, while its minimum bactericidal/fungicidal concentration ranged from 3.125 mg/ml to 200. Based on the present research, the essential oil Citrus aurantium antioxidant potential and antimicrobial activity on several food-borne pathogens tested and thus can be a good source of food industrial.