Brevipalpus yothersi Baker (Tenuipalpidae) development in sweet orange plants is influenced by previous mite infestation and the presence of shelters.

Citrus leprosis is the most important viral disease affecting citrus. The disease is caused predominantly by CiLV-C and is transmitted by Brevipalpus yothersi Baker mites. This study brings some insight into the colonization of B. yothersi in citrus [(Citrus × sinensis (L.) Osbeck (Rutaceae)] previously infested by viruliferous or non-viruliferous B. yothersi. It also assesses the putative role of shelters on the behavior of B. yothersi. Expression of PR1 and PR4 genes, markers of plant defense mechanisms, were evaluated by RT-qPCR to correlate the role of the plant hormonal changes during the tri-trophic virus-mite-plant interplay. A previous infestation with either non-viruliferous and viruliferous mites positively influenced oviposition and the number of adult individuals in the resulting populations. Mite populations were higher on branches that had received a previous mite infestation than branches that did not. There was an increase in the expression of PR4, a marker gene in the jasmonic acid (JA) pathway, in the treatment with non-viruliferous mites, indicating a response from the plant to their feeding. Conversely, an induced expression of PR1, a marker gene in the salicylic acid (SA) pathway, was observed mainly in the treatment with viruliferous mites, which suggests the activation of a plant response against the pathogen. The earlier mite infestation, as well as the presence of leprosis lesions and a gypsum mixture as artificial shelters, all fostered the growth of the B. yothersi populations after the second infestation, regardless of the presence or absence of CiLV-C. Furthermore, it is suggested that B. yothersi feeding actually induces the JA pathway in plants. At the same time, the CiLV-C represses the JA pathway and induces the SA pathway, which benefits the mite vector.

Impact of diseases and pests on premature fruit drop in sweet orange orchards in São Paulo state citrus belt, Brazil.

BACKGROUND: Despite technical improvements in the citrus chain and leadership in orange production achieved in the past decades, premature fruit drop remains a major component of crop loss in São Paulo state citrus belt, the largest sweet orange production area in the world. The present study aimed to determine, during five consecutive seasons, the impact of the diseases and pests on premature fruit drop in the orange belt. RESULTS: Fruit drop due to the main diseases and pests averaged approximately 11.0%, which corresponded to approximately 63% of the annual fruit drop. The average fruit drop rate due to fruit borer and fruit flies combined was 4.0%, Huanglongbing (HLB) 3.3%, black spot 2.6%, leprosis 1.0% and citrus canker 0.3%. The average amount of fruit drop (million 40.8 kg boxes) and value of crop losses (million US$ dollars), in five seasons, were 12.7 and 66.2 for fruit borer/fruit flies, 11.0 and 57.9 for HLB, 8.1 and 42.2 for black spot, 3.1 and 15.6 for leprosis, and 0.9 and 4.9 for citrus canker, respectively. CONCLUSION: Fruit borer and fruit flies (combined), HLB, black spot, leprosis and citrus canker are, in this order, the main diseases and pests in the orange belt of São Paulo state. All of these causes significantly increased the overall fruit drop rate in the evaluated seasons. The results will contribute to the development of the Brazilian citrus industry, while showing to other citrus-growing regions the potential that diseases and pests have to jeopardize production. © 2022 Society of Chemical Industry.

Large scale survey of yeasts in frozen concentrated orange juice (FCOJ): Occurrence, diversity, and resistance to peracetic acid.

This study aimed to evaluate the occurrence and diversity of yeasts in frozen concentrated orange juice (FCOJ) and assess the resistance of yeasts to peracetic acid. One thousand five hundred (n = 1500) samples of frozen concentrated orange juice (FCOJ) were analyzed, and 280 yeast strains were isolated and identified. Candida represented 37% of all isolates, and the main species identified were Candida pseudointermedia and C. orthopsilosis. Other yeasts identified were Starmerella, Wickerhamiella, Wickerhamiella, Clavispora, Kodamaea, Meyerozyma, Rhodotorula, Trichosporon, Wickerhamomyces, Kluyveromyces, Hanseniaspora, Saccharomyces, Torulaspora, and Zygosaccharomyces. The exogenous origin of the contamination in FCOJ samples analyzed was shown by the high diversity, corroborated by the Simpson (D) and Shannon (H') indices. From a total of 227 yeasts strains tested, more than 20% were able to withstand peracetic acid concentrations >200 ppm, with emphasis on W. anomalus (300 ppm), W. sergipensis (350 ppm), C. rugopelliculosa (350 ppm), K. marxianus (450 ppm), C. parapsilosis (500 ppm), C. pseudointermedia (500 ppm), W. sorbosivorans (500 ppm), C. boleticola (600 ppm), S. cerevisiae (700 ppm) and C. orthopsilosis (750 ppm). This study adds novel data regarding the occurrence and diversity of yeasts present in FCOJ and their resistance to a chemical compound commonly employed in the sanitization of processing and distribution premises and vehicles. These findings are essential to support the development of measures for proper mitigation of contamination of orange juice towards reducing the risks of spoilage by yeasts during FCOJ transportation/storage or when FCOJ is used as an ingredient.

Spatiotemporal association between the mite Brevipalpus yothersi and Citrus leprosis virus C in orange orchards.

Citrus leprosis virus C (CiLV-C) is an economically important pathogen and the main causative agent of leprosis disease in citrus orchards. The main vector of this disease, the mite Brevipalpus yothersi, is widely distributed in Mexican orchards on a wide range of citrus species. Despite the importance of both the virus and the mite, field studies recording their occurrence and co-occurrence are practically non-existent. We systematically sampled orange orchards for both CiLV-C and B. yothersi throughout the year. The distribution of the CiLV-C and B. yothersi was evaluated on each sampling occasion and their spatiotemporal associations were determined. Specifically, 100-112 orange trees, distributed in 18 rows (five or six trees per row), were sampled monthly between March 2017 and February 2018 (11 sampling dates). Twenty leaves per tree were sampled on each occasion. The number of mites per tree and the percentage of leaves per tree with disease symptoms were recorded. On each sampling occasion, spatiotemporal associations between mites and disease were determined using the Spatial Analysis by Distance Indices (SADIE) method. CiLV-C and B. yothersi were identified using molecular methods. Throughout the study, the distribution of CiLV-C was aggregated and the distribution of B. yothersi was random. No association was found between the virus and the mite on any of the sampling dates. In total, 173 mites were collected, but only 43 mites were found to be carrying CiLV-C. The reason for this lack of association between the virus and the mite, as well as the impact of our findings on the epidemiology of the disease in orange orchards, are discussed.

The influence of citrus rootstocks on the relationship between the mite Brevipalpus phoenicis and citrus leprosis disease.

BACKGROUND: Leprosis is one of the most serious citrus plant diseases. Leprosis-affected plants, especially sweet orange [Citrus sinensis (L.) Osbeck], which is the most widely cultivated citrus fruit worldwide, show reduced photosynthetic capacity and severe defoliation. The aim was to evaluate the relationship between the Brevipalpus phoenicis (Geijskes) vector mite and citrus leprosis disease in Pera sweet orange plants grafted on different rootstocks. Data were analysed using numerical classification and conventional statistical analysis (ANOVA). RESULTS: Both viruliferous and non-viruliferous B. phoenicis populations increased in number on plants maintained at low soil water content. Among the evaluated rootstocks, Sunki mandarin proved least favourable for mite population increase. Furthermore, the viruliferous mite population increased more rapidly than the non-viruliferous mite population. CONCLUSION: The Cleopatra rootstock showed low variability in leaf nitrogen content, low mite number and low leprosis severity, thereby producing the most favourable results. Under appropriate moisture conditions, the Sunki rootstock produced the best results, showing the least severe leprosis. Rangpur lime rootstock grown in soil with varying water content showed the greatest variation in foliar nitrogen content, mite number and severity of leprosis and is considered to be the most difficult rootstock to manage.

Comparative morpho-anatomical studies of the lesions caused by citrus leprosis virus on sweet orange.

The leprosis disease shows a viral etiology and the citrus leprosis virus is considered its etiologic agent. The disease may show two types of cytopatologic symptom caused by two virus: nuclear (CiLV-N) and cytoplasmic (CiLV-C) types. The aim of this study was to compare the morpho-anatomical differences in the lesions caused by leprosis virus-cytoplasmic and nuclear types in Citrus sinensis (L.) Osbeck 'Pêra'. Leaf and fruit lesions were collected in Piracicaba/São Paulo (cytoplasmic type) and Monte Alegre do Sul/São Paulo and Amparo/São Paulo (nuclear type). The lesions were photographed and then fixed in Karnovsky solution, dehydrated in a graded ethylic series, embedded in hydroxy-ethyl methacrylate resin (Leica Historesin), sectioned (5 microm thick), stained and mounted in synthetic resin. The digital images were acquired in a microscope with digital video camera. Leaf and fruit lesions caused by the two viruses were morphologically distinct. Only the lesion caused by CiLV-N virus presented three well-defined regions. In both lesions there was the accumulation of lipidic substances in necrotic areas that were surrounded by cells with amorphous or droplets protein. Only leaf and fruit lesions caused by CiLV-N virus exhibited traumatic gum ducts in the vascular bundles.

Comparative morpho-anatomical studies of the lesions caused by citrus leprosis virus on sweet orange

The leprosis disease shows a viral etiology and the citrus leprosis virus is considered its etiologic agent. The disease may show two types of cytopatologic symptom caused by two virus: nuclear (CiLV-N) and cytoplasmic (CiLV-C) types. The aim of this study was to compare the morpho-anatomical differences in the lesions caused by leprosis virus-cytoplasmic and nuclear types in Citrus sinensis (L.) Osbeck 'Pêra'. Leaf and fruit lesions were collected in Piracicaba/São Paulo (cytoplasmic type) and Monte Alegre do Sul/São Paulo and Amparo/São Paulo (nuclear type). The lesions were photographed and then fixed in Karnovsky solution, dehydrated in a graded ethylic series, embedded in hydroxy-ethyl methacrylate resin (Leica Historesin), sectioned (5 μm thick), stained and mounted in synthetic resin. The digital images were acquired in a microscope with digital video camera. Leaf and fruit lesions caused by the two viruses were morphologically distinct. Only the lesion caused by CiLV-N virus presented three well-defined regions. In both lesions there was the accumulation of lipidic substances in necrotic areas that were surrounded by cells with amorphous or droplets protein. Only leaf and fruit lesions caused by CiLV-N virus exhibited traumatic gum ducts in the vascular bundles.

A doença leprose dos citros tem etiologia viral sendo o citrus leprosis virus seu agente etiológico. Demonstrou-se que há dois vírus distintos que causam sintomas de leprose em ci-tros: citoplasmático (CiLV-C) e o nuclear (CiLV-N). O objetivo desse estudo foi comparar as diferenças morfo-anatômicas nas lesões causadas por CiLV-C e por CiLV-N em laranjeira doce (Citrus sinensis (L.) Osbeck) 'Pêra'. As lesões foliares e dos frutos foram coletadas em Piracicaba/SP (tipo citoplas-mático) e em Monte Alegre do Sul/SP e Amparo/SP (tipo nuclear). As lesões foram fotografadas e em seguida fixadas em solução Karnovsky, desidratadas em série etílica, incluídas em historesina e secionadas em micrótomo rotativo. As lâminas foram coradas, analisadas e fotografadas. As lesões foliares e do fruto causadas pelos dois vírus eram morfologicamente distintas. Somente a lesão causada por CiLV-N apresentou três regiões bem definidas. Em ambas as lesões ocorreu acúmulo de substâncias lipídicas nas áreas necrosadas que se achavam envoltas por células com conteúdo protéico amorfo ou em gotas. Somente as lesões da folha e do fruto causadas pelo CiLV-N exibiram ductos traumáticos gomosos nos feixes vasculares.

Application of whole genome amplification and quantitative PCR for detection and quantification of spoilage yeasts in orange juice.

Small cell numbers in complex food matrices and undefined PCR inhibitors often limit detection and identification of DNA species by molecular techniques. Thus in many industrial situations enrichment growths are performed. However, growth speed of different species in complex microbial mixtures in defined media is in most cases different, thus final results do not always reflect the starting situation. We tested DNA-strand displacement whole genome amplification as a possible substitute of enrichment growth. Using whole genome amplification on orange juice contaminated with Saccharomyces cerevisiae, we lowered detection level from 10(6) down to 10(2) cfu/ml. Whole genome amplification showed to be linear (R=0.992) and the relative yeast DNA copy number compared to other DNA templates did not change thus allowing quantitative estimation of initial contamination by quantitative PCR. Using melting point analysis, we were able to distinguish between the PCR products of the 5.8S-ITS region, obtained with universal primers from pure cultures of S. cerevisiae and Hanseniaspora uvarum, two major spoilage yeasts in orange juice and forming part of wine microbiota during fermentation. However, in mixed-contaminated samples, identification of both species was hampered by preferential appearance of the melting peak coinciding with H. uvarum, except when S. cerevisiae was the dominating species. Application of whole genome amplification did not prevent the preferential detection of H. uvarum. This handicap was resolved by applying an enrichment procedure up to saturation after which the melting peak of both species could clearly be identified.

The complete nucleotide sequence and genomic organization of Citrus Leprosis associated Virus, Cytoplasmatic type (CiLV-C).

The Citrus leprosis disease (CiL) is associated to a virus (CiLV) transmitted by Brevipalpus spp. mites (Acari: Tenuipalpidae). CiL is endemic in Brazil and its recently spreading to Central America represents a threat to citrus industry in the USA. Electron microscopy images show two forms of CiLV: a rare nuclear form, characterized by rod-shaped naked particle (CiLV-N) and a common cytoplasmic form (CiLV-C) associated with bacilliform-enveloped particle and cytoplasmic viroplasm. Due to this morphological feature, CiLV-C has been treated as Rhabdovirus-like. In this paper we present the complete nucleotide sequence and genomic organization of CiLV-C. It is a bipartite virus with sequence similarity to ssRNA positive plant virus. RNA1 encodes a putative replicase polyprotein and an ORF with no known function. RNA2 encodes 4 ORFs. pl5, p24 and p61 have no significant similarity to any known proteins and p32 encodes a protein with similarity to a viral movement protein. The CiLV-C sequences are associated with typical symptoms of CiL by RT-PCR. Phylogenetic analysis suggests that CiLV-C is probably a member of a new family of plant virus evolutionarily related to Tobamovirus.

Pharmacokinetics and relative bioavailability of clofazimine in relation to food, orange juice and antacid.

BACKGROUND: Clofazimine is potentially useful for the treatment of disease due to multidrug resistant Mycobacterium tuberculosis, as well as leprosy and certain chronic skin diseases. Its pharmacokinetics have been incompletely characterized. This study was conducted to explore issues relating to bioavailability in the presence of food, orange juice, and antacid. METHODS: A 5 drug regimen consisting of clofazimine, cycloserine, ethionamide, para-aminosalicyclic acid, and pyridoxime was administered to healthy subjects four times using a four period cross-over design with two weeks washout between treatments. Subjects also received orange juice, a high fat meal, aluminum/magnesium antacid, or only water in random order with the drug regimen. The pharmacokinetics of clofazimine were assessed using individual- and population-based methods and relative bioavailability compared to fasting administration was determined. RESULTS: Clofazimine exhibited a sometimes prolonged and variable lag-time and considerable variability in plasma concentrations. From the population analysis (one-compartment model), the mean oral clearance was 76.7 l/h (CV=74.2%) and mean apparent volume of distribution was 1470 l (CV=36.3%). The first-order absorption rate constant ranged from 0.716 to 1.33 h(-1) (pooled CV=61.7%). Residual (proportional) error was 49.1%. Estimates of bioavailability compared to fasting administration were 145% (90% CI, 107-183%) for administration with high fat food, 82.0% (63.2-101%) for administration with orange juice, and 78.5% (55.1-102%) for administration with antacid. CONCLUSION: Administration of clofazimine with a high fat meal provides the greatest bioavailability, however, bioavailability is associated with high inter- and intra-subject variability. Both orange juice and aluminum-magnesium antacid produced a reduction in mean bioavailability of clofazimine.