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Citrus tristeza virus (CTV) in Texas was first reported in the 1950s and has since been sporadically reported in the residential areas in the Upper Gulf Coast region. Because the major rootstock for commercial citriculture in South Texas is sour orange, which is susceptible to CTV decline, the spread of CTV into South Texas can pose a great threat to Texas citrus industry. Thirty-six CTV-positive samples, collected during surveys conducted in the Upper Gulf Coast area of Texas from 2013 to 2018, were first analyzed by strain-specific real-time PCR (RT-PCR) targeting various regions of CTV Open reading frame (Orf) 1a and then by amplicon sequencing derived from p25 and p20 region of CTV genome. Among 36 samples, 33 were successfully genotyped by strain-specific RT-PCR and by amplicon sequencing followed by phylogenetic analysis. Variability in the detection of CTV strains was observed over a 6-year period. In 2013, T3 and T30 were the only strains detected in the Upper Gulf Coast of Texas, but in further surveys until 2018, additional strains were detected, including T36, VT, and RB. Mixed infections were also detected in 14 samples comprising about 42% of CTV samples examined in the study. Although genotyping mixed infection samples by targeting Orf 1a and full-length p25, residing in the 5′ and 3′ region of the CTV genome, respectively, confirmed the presence of multiple strains in these samples, incongruent genotyping data were observed. These findings suggested that the current status of CTV strain diversity in Texas Upper Gulf Coast region might have been established by multiple introductions of CTV-infected plant materials for propagation and with a potential recombination in planta.

Citrus canker caused by Xanthomonas citri subsp. citri (syn. X. axonopodis pv. citri) was first reported in South Texas during the early 1900s, prompting quarantine and eradication programs by the federal government. Its last reported incidence in Texas was from two trees from Corpus Christi in 1943. In May 2016, USDA Animal and Plant Health Inspection Service-Plant Protection and Quarantine (PPQ) confirmed the presence of the Asiatic A strain, which is by far the most widespread and severe form infecting most citrus varieties, on two sour orange trees in a park in Houston. Subsequently, infected trees were detected in a nursery in Richmond (Fort Bend Co.) in August 2016 and in dooryard trees in Pearland (Brazoria Co.) in 2018. PPQ and Texas Department of Agriculture collected a combined 1,949 positive A strain samples from Harris, Fort Bend, and Brazoria Counties during May 2016 to January 2021. These findings resulted in seven active citrus canker quarantine zones being established in the Upper Gulf Coast area. The Texas Department of Agriculture is removing positive trees, and surveys are continuing.

Seeds from four citrus rootstocks including sour orange, Bitters-C22 citrandarin, Sarawak pummelo Rio Red grapefruit, and Sarawak pummelo Bower mandarin were exposed to high inoculum levels of Phytophthora nicotianae to screen for tolerance. Inoculation of pregerminated seeds (PGIS) and non-PGIS was carried out. The average P. nicotianae propagule counts from the soil samples where these seedlings were raised ranged from 424 to 1361 colony forming units/cm3. The proportion of live to dead plants was recorded at 11 months postinoculation, which showed that Sarawak Bower performed significantly better than other rootstocks. Evaluation of the rootstocks 18 months postinoculation resulted in only one surviving sour orange plant, which suggests potential rootstock resistance.

Huanglongbing (HLB, citrus greening disease) in the major citrus-producing states of the United States is associated with Candidatus Liberibacter asiaticus (CLas), which is vectored by the Asian citrus psyllid (ACP). Surveys were conducted in Texas from 2007 to 2017 to assess the prevalence and titer of CLas in ACPs and citrus trees. ACP and citrus leaf tissue samples were collected from suspect trees in residential areas and commercial groves (orchards) and assayed for CLas by quantitative PCR. CLas detection in ACPs (2011) preceded that of citrus trees (2012) by several months. Annual incidences of CLas-positive ACPs and leaf tissue followed an exponential growth pattern over the survey period, varying from 0.03 to 28.7% in ACPs and 0.6 to 36.5% in citrus trees. There was a significant and positive relationship between the monthly incidences of CLas-positive ACP and leaf tissue samples. The proportion of HLB detection sites also increased with time, reaching 26 and 40% of commercial groves and residential sites, respectively, by 2017. Seasonal variations were observed in the incidences of CLas-positive ACPs and citrus trees such that significantly more CLas-positive ACPs and trees were recorded during the fall and winter of a given year relative to the hot summer. A temporal analysis of the class distribution of cycle threshold values revealed a trend of increased bacterial accumulation in ACPs and trees over time, with the trend more pronounced for the former than the latter host type. These findings provide a comprehensive insight into the ongoing CLas/HLB epidemic in Texas, with potential lessons for California and other citrus-producing areas where the disease is not yet established.

Plant diseases contribute to an estimated annual crop loss of $60 billion worldwide, threatening the food security and thereby the survival of humankind. Plant pathogens are an important component of the widely accepted disease triangle concept where they cause diseases in susceptible hosts under favorable environmental conditions. Under such favorable environmental conditions, pathogens often spread rapidly to new hosts. Therefore, early and accurate detection of a disease and diagnosis of its causal agent is highly important for disease control and sustainable agricultural production. This chapter provides information on an array of methods actively being used to detect and diagnose diseases. We include discussion of established methods such as microscopy and serology, indexing, and PCR as well as methods that are actively under development. An elaborate account of information is provided on promising diagnostic methods based on programmable nucleic acid-binding proteins such as clustered regularly interspaced palindromic repeat (CRISPR)-associated (CAS) proteins, zinc finger (ZnF) proteins, and transcription activator-like effector (TALE) proteins. Finally, we discuss indirect pathogen detection technologies that utilize optical sensors to identify changes in the host plant properties due to a disease.

Huanglongbing (HLB), or Citrus Greening, is one of the most devastating diseases affecting agriculture today. Widespread throughout Citrus growing regions of the world, it has had severe economic consequences in all areas it has invaded. With no treatment available, management strategies focus on suppression and containment. Effective use of these costly control strategies relies on rapid and accurate identification of infected plants. Unfortunately, symptoms of the disease are slow to develop and indistinct from symptoms of other biotic/abiotic stressors. As a result, diagnosticians have focused on detecting the pathogen, Candidatus Liberibacter asiaticus, by DNA-based detection strategies utilizing leaf midribs for sampling. Recent work has shown that fibrous root decline occurs in HLB-affected trees before symptom development among leaves. Moreover, the pathogen, Ca. Liberibacter asiaticus, has been shown to be more evenly distributed within roots than within the canopy. Motivated by these observations, a longitudinal study of young asymptomatic trees was established to observe the spread of disease through time and test the relative effectiveness of leaf- and root-based detection strategies. Detection of the pathogen occurred earlier, more consistently, and more often in root samples than in leaf samples. Moreover, little influence of geography or host variety was found on the probability of detection.

Phytophthora-induced foot rot, also known as gummosis, is an important disease affecting citrus production worldwide. In Texas, the third-largest citrus-producing state in the United States, limited information is available on the etiology and epidemiology of foot rot in commercial orchards. This study comprises a survey of foot rot incidence and severity in Texas and the characterization of Phytophthora isolates associated with the disease. Surveys in 2015 and 2017 of 30 orchards in the Lower Rio Grande Valley (LRGV) region where commercial citrus production is concentrated in the state revealed that foot rot occurred in 97% of the orchards assessed. Overall, foot rot symptoms were observed on 33.7% of the trees evaluated and the disease severity index in the region was rated at 14.2 and 16.5% in 2015 and 2017, respectively. Lesions were mostly present on the scion, while the rootstock (sour orange) was not affected. Phytophthora nicotianae was the only Phytophthora sp. isolated from the surveyed orchards and from five additional residential sites on the Texas Coastal Bend (TCB). Sporangia and chlamydospores from 34 representative LRGV isolates of P. nicotianae were larger than those of TCB isolates. In both LRGV and TCB, A1 and A2 mating types were present in the same location, albeit the A2 mating type was more prevalent. All isolates were sensitive to mefenoxam (50% inhibition in the presence of mefenoxam [EC50] < 0.5 µg/ml), except for one TCB isolate (EC50 = 143.6 µg/ml). Our research indicates that treatment for Phytophthora foot rot in the region is necessary and, although mefenoxam is still useful, alternating chemistries for resistance management are required.

Huanglongbing (HLB, citrus greening disease) in the major citrus-producing states of the United States is associated with

Citrus fibrous root tissue was evaluated as an alternative source material for huanglongbing (HLB) diagnosis by real-time PCR using primer-probe set TXCChlb, developed in the present study based on 16S rDNA of “Candidatus Liberibacter asiaticus” (CLas). Real-time PCR data obtained with DNA samples prepared from leaf and fibrous root tissue collected from mature (> 10 years old) and young (4–5 years old) citrus trees growing in Texas and Florida confirmed that root HLB test is more sensitive than the leaf HLB test in terms of the detection rate of HLB positive trees. In addition, the current study confirmed that HLB can be diagnosed at the pre-symptomatic stage using the root HLB test to facilitate efficient removal of HLB-positive, asymptomatic trees that could serve as a source for HLB. The new HLB diagnostic method using root tissue described in the current study can assist in deploying a more efficient disease management strategy to deter HLB spread, especially at the pre-symptomatic stage.

In October 2015, a Mexican lime exhibiting citrus canker symptoms was found by the USDA APHIS PPQ in a residential property in Rancho Viejo, Cameron County, Texas. Real-time PCR analysis detected the presence of Xanthomonas citri subsp. citri; USDA APHIS PPQ in Beltsville, MD confirmed this diagnosis. A delimiting survey was initiated and suspect leaf samples were collected and sent to the PPQ Beltsville lab for analysis. By October 2017, leaf samples from 197 trees were confirmed positive for citrus canker, all within a 5-mile radius; in addition, a further 59 symptomatic trees were found and all 256 infected trees (254 Mexican lime, one makrut lime, and one Ponderosa lemon) were removed. Survey data collected on stem lesions suggested the oldest lesions to be between 4 to 6 years old. A host-range study using 12 citrus varieties, including the major commercial varieties grown in Texas, were inoculated with crude leaf extracts from symptomatic leaves by leaf infiltration method. Mexican limes and alemow were the only citrus plants that developed definitive canker lesions. A combination of host range, serological, and molecular tests suggested that this isolate was different from the typical Asiatic strain and more similar to X. citri subsp. citri AW which was only previously reported from Florida on Mexican lime and alemow.

Detection of ‘Candidatus Liberibacter asiaticus’ represents one of the most difficult, yet critical, steps of controlling Huanglongbing disease. Efficient detection relies on understanding the underlying distribution of bacteria within trees. To that end, we studied the distribution of ‘Ca. L. asiaticus’ in leaves of ‘Rio Red’ grapefruit trees and in roots of ‘Valencia’ sweet orange trees grafted onto sour orange rootstock. We performed two sets of leaf collection on grapefruit trees; the first a selective sampling targeting symptomatic leaves and their neighbors and the second a systematic collection disregarding symptomology. From uprooted orange trees, we exhaustively sampled fibrous roots. In this study, the presence of ‘Ca. L. asiaticus’ was detected in leaves using real-time polymerase chain reaction (PCR) targeting the 16S ribosomal gene and in roots using the rpIJ/rpIL ribosomal protein genes and was confirmed with conventional PCR and sequencing of the rpIJ/rpIL gene in both tissues. Among randomly collected leaves, ‘Ca. L. asiaticus’ was distributed in a patchy fashion. Detection of ‘Ca. L. asiaticus’ varied with leaf symptomology with symptomatic leaves showing the highest frequency (74%) followed by their neighboring asymptomatic leaves (30%), while randomly distributed asymptomatic leaves had the lowest frequency (20%). Among symptomatic leaves, we found statistically significant differences in mean number of bacterial cells with respect to both increasing distance of the leaf from the trunk and cardinal direction. The titer of ‘Ca. L. asiaticus’ cells was significantly greater on the north side of trees than on the south and west sides. Moreover, these directions showed different spatial distributions of ‘Ca. L. asiaticus’ with higher titers near the trunk on the south and west sides as opposed to further from the trunk on the north side. Similarly, we found spatial variation in ‘Ca. L. asiaticus’ distribution among root samples. ‘Ca. L. asiaticus’ was detected more frequently and bacterial abundances were higher among horizontally growing roots just under the soil surface (96%) than among deeper vertically growing roots (78%). Bacterial abundance declined slightly with distance from the trunk. These results point to paths of research that will likely prove useful to combating this devastating disease.

Yeasts from all immature life stages of Mexican fruit fly Anastrepha ludens (Loew) (Diptera: Tephritidae) from diet, insectary air, and rearing materials were isolated, identified and evaluated for pathogenicity. Fifteen species of yeasts with one to genus level were identified from 72 yeast cultures obtained. Zygosaccharomyces bailii was the only yeast found to be highly pathogenic to Mexican fruit fly. Seventy-two hours post inoculation, the diet in bioassay cups with Z. bailii consistently showed signs of fermentation with gas bubbling causing the migration of larvae to the walls and lids of bioassay cups. The spent diet from Z. balii-infested cups was crusty, cracked and had a pasty layer. Many larvae were small, moribund, and discolored, appearing caramel or blackish. Insect yield loss with Z. bailii in comparison to that of control ranged from 10 to 44% for larvae and 14 to 47% for pupae. Additionally, Z. bailii caused a reduction in mean pupal weight. The weakly pathogenic yeasts produced significantly less yield of larvae and pupae than the nonpathogenic ones included Trichosporon montevideense, Clavispora lucitaniae, Candida sp., C. rugosa, and Rhodotorula mucilaginosa. Yield loss of larvae caused by this group ranged from 12 (C. lusitaniae) to 18% (R. mucilaginosa). Yield losses for pupa were similar to that of larvae. The mean pupa weight for these species was above the minimum acceptable (16.50 mg) for the SIT program. The nonpathogenic yeast produced yields of larvae and pupae similar to the control included Cryptococcus diffluens, Pichia kudriavzevii, Meyerozyma guilliermondii, Wickerhamomyces anomalus, Trichosporon asahii, Debaryomyces hansenii, Candida tropicalis, Cryptococcus sp., Candida parapsilosis, and Hanseniaspora opuntiae. In conclusion, the identification and management of insect pathogenic yeasts, such as Z. balii in mass rearing systems of Mexican fruit fly must be considered to avoid their potential negative effects.

Sweet orange scab (SOS) is a fungal disease of citrus which is caused by Elsinoe australis. It affects the aesthetics of the fruit by forming wart-like protruded lesions on the fruit skin, and also affects the leaves which act as source of inoculum in the orchards. SOS is widespread in the different citrus species such as grapefruit (Citrus x paradisi Macfd.), sweet orange [C. sinensis (L.) Osb.], and mandarin (C. reticulata Blanco). In this study, we report the development of symptoms and changes in polyphenolic profile of ‘Rio Red’ grapefruit leaves in response to E. australis infection. Qualitative and quantitative analysis of polyphenols in ‘Rio Red’ grapefruit leaves inoculated with E. australis were conducted using high performance liquid chromatography (HPLC) at two different time points post inoculation. Development of SOS symptoms under light was recorded and categorized into four different morphological stages. Caffeic acid, luteolin-7-O glycoside, naringin, naringenin, apigenin-7-O glycoside and eriodictoyl were identified in healthy grapefruit leaves. The quantitative changes in the identified polyphenols were measured 9 days post-inoculation by comparing levels on the 9-day control versus the 9-day post-inoculated leaves. Total polyphenol levels decreased substantially in E. australis-infected ‘Rio Red’ grapefruit leaves 9 days post-inoculation. Levels of naringin, naringenin, apigenin glucoside and eriodictoyl decreased with E. australis infections indicating the pathogen’s ability to overcome some of plant’s defenses. Compared to 9-day controls, 9-day inoculated leaves had much less caffeic acid.

Zebra chip disease of potato is caused by the bacterial pathogen ‘Candidatus Liberibacter solanacearum’ and is a growing concern for commercial potato production in several countries in North and Central America and New Zealand. ‘Ca. L. solanacearum’ is vectored by the potato psyllid Bactericera cockerelli, which transmits the pathogen to several cultivated and wild solanaceaous host plants. Silverleaf nightshade (SLN), Solanum elaeagnifolium, is a common weed in the Lower Rio Grande Valley of Texas and a host for both the potato psyllid and ‘Ca. L. solanacearum’. SLN plants were successfully inoculated with ‘Ca. L. solanacearum’ under laboratory conditions. Retention studies demonstrated that ‘Ca. L. solanacearum’-infected SLN planted in the field in January 2013, concurrent with commercial potato planting, retained the pathogen under field conditions throughout the year despite extensive dieback during summer. The presence of ‘Ca. L. solanacearum’ was confirmed in leaves, roots, and stolons of SLN plants collected the following year using polymerase chain reaction. Acquisition assays using B. cockerelli adults also revealed that SLN retained the pathogen. Transmission studies determined that B. cockerelli can acquire ‘Ca. L. solanacearum’ within a 2-week acquisition access period on ‘Ca. L. solanacearum’-infected SLN and subsequently transmit the pathogen to potato. These results demonstrate that SLN plants can serve as a reservoir for ‘Ca. L. solanacearum’, providing a source of inoculum for B. cockerelli adults colonizing potato the next season. The presence of SLN plants all year round in the LRGV makes the weed an epidemiologically important host. These findings underscore the importance of eradicating or managing SLN plants growing in the vicinity of potato fields to prevent spread of ‘Ca. L. solanacearum’ and damage caused by zebra chip.

Huanglongbing (HLB), also referred to as citrus greening disease has caused significant losses to the citrus in- dustry in the United States and elsewhere. In our previous studies, we observed the fluctuation of some primary and sec- ondary metabolites in response to biotic (psyllids feeding that transfers bacteria; i.e., Candidatus Liberibacter spp. that cause greening disease) and abiotic (water deficit) stress factors in citrus. In the current report, we evaluated the changes in polyphenolic compounds in Satsuma leaves in response to Asian citrus psyllid feeding stress and the water stress. In general, polyphenolic levels increased in Satsuma leaves in response to insect and water stress. Specifically, polyphenols such as chlorogenic acid, rutin, diosmin, luteolin7-O-glucoside, and narangin levels increased significantly in response to both biotic and abiotic stress. On the other hand, while caffeic acid levels significantly increased in water stressed plants, their levels drastically declined to the level of being undetectable in leaves stressed by psyllid feeding. Differences be- tween the two types of stresses were also observed in the levels of apigenin 7-O-glucoside where it decreased significantly in water stressed leaves but not in leaves stressed by psyllid feeding; i.e. changes in the levels of apigenin 7-O-glucoside and caffeic acid were opposite in response to water or feeding stresses. Hesperidin levels were not affected by the water stress or by psyllid feeding. The findings may help to better understanding plant psyllid interactions and may be helpful in developing effective management practices to control the spread of citrus greening disease.

a b s t r a c t Fibrous root samples from Huanglongbing symptomatic (S) and asymptomatic (AS) ‘Valencia’ sweet orange trees (Citrus sinensis) grafted onto sour orange (C. aurantium) rootstock were analyzed using different DNA extraction procedures and primers to detect Candidatus Liberibacter asiaticus. Root sampling procedure was modified from the conventional use of soil probe to reduce fatigue, and to facilitate sample collection. DNA extraction kits including Qiagen DNeasy, Mo Bio PowerSoil and PowerPlant generated root DNA suitable for polymerase chain reaction (PCR) diagnostic assays. Quantitative PCR (qPCR) using HLBaspr primers and probe on root DNA extracts from a total of 206 samples from 60 AS trees showed unreliable results for the presence of Ca. L. asiaticus. Except for one sample, all the remaining samples gave negative qPCR result using LJ900fpr primers and probe. Further analysis of roots samples from S and AS trees indicate that HLBaspr is not suitable for Ca. L. asiaticus diagnosis in root samples. Leaf samples from all 60 AS trees showed negative qPCR result with HLBaspr. Conventional PCR (cPCR) using OI1/OI2c primers on 10 randomly selected root samples, from the 206 samples, produced non-specific amplification products which were easily distinguished from products specific to Ca. L. asiaticus seen in the positive control. The same root samples did not produce any amplification product in cPCR using A2/J5 primers. Any of the above mentioned cPCR or qPCR assays could accurately detect Ca. L. asiaticus in S leaf samples of Ca. L. asiaticus-infected trees. Root samples from 10 (5 S and 5 AS) additional trees analyzed by cPCR using A2/J5 and Las606/LSS primers and qPCR using LJ900fpr and CQULA (CQULA04F/CQULA04R, and TaqMan probe CQULAP10) clearly showed that all these primers are efficient in detecting Ca. L. asiaticus in root samples. There was no difference in frequency and consistency of Ca. L. asiaticus detection for root samples collected near the tree trunk compared to the roots collected about 1.8 m from the trunk. Ca. L. americanus was not detected in either root or leaf samples.

The effects of biotic and abiotic stresses on changes in amino acids and polyamine levels in Satsuma orange (Citrus unshiu; cultivar Owari) leaves were investigated. Asian citrus psyllids Diaphorina citri (Kuwayama) (ACP) infestation was used to induce biotic stress while a water deficit was imposed to induce abiotic stress. Potted trees were infested by placing 50 psyllids on 3 citrus leaves enclosed in nylon mesh bags for 5 d. A parallel set of plants were kept water stressed by maintaining the soil at 20% water holding capacity for 5 d. Levels of total free amino acids were higher in water stressed and ACP infested leaves. Polyamine putrescine increased in infested leaves but not in water stressed leaves. Proline was the most abundant amino acid and its levels significantly increased by both biotic and abiotic stresses. Proline levels in infested leaves were significantly higher than the water stressed leaves. Histidine, methionine, asparagine, arginine, serine, and leucine levels also increased significantly in infested leaves, but in water stressed leaves only leucine, methionine, and threonine increased. Levels of amino acids, such as tyrosine, isoleucine, phenylalanine, glutamic acid, and alanine, declined in infested leaves. Under water stress asparagine, phenylalanine, serine, and histidine also declined compared to controls. This indicates that while proteolysis occurred under both stresses, metabolic conversion of amino acids was different under the two stresses. In ACP infested leaves some amino acids may be used as feeding material and/or converted into secondary metabolites for defense.

Huanglongbing (HLB) disease, associated with ‘Candidatus Liberibacter asiaticus’ (CLas), is primarily spread via infected citrus nursery trees and by infective Asian citrus psyllid, the insect vector. Recently, the Texas Department of Agriculture initiated regulations requiring commercial and retail citrus nurseries in Texas to transition from traditional open-field to enclosed facilities with insect-resistant screens to mitigate the risk of nurseries serving as sources of CLas. Although several nursery production facilities have adopted this regulation, non-enclosed nurseries persist and pose a significant threat to the citrus industry as potential sources of CLas. A systematic survey for HLB was embarked on in a semi-open nursery facility in South Texas in April 2014. Leaf tissue samples taken from 94 trees representing 5% of the total number of potted trees in the nursery were tested for CLas by quantitative and conventional PCR assays. Of 94 trees tested, 3.2% (3 trees) were positive for CLas by both assays. The presence of CLas in the PCR-positive samples was confirmed by multi-locus sequence analyses. The results represent the first report of HLB in a nursery facility in Texas, and underscore the need for more intensive surveillance for HLB in citrus nursery stock as an integral component of HLB mitigation efforts in Texas. Accepted for publication 27 August 2014. Published 15 December 2014.

The Asian citrus psyllid, Diaphorina citri Kuwayama, one of the known vectors for citrus greening disease or Huanglongbing (HLB) pathogens, has been present in Texas for over a decade, but the detection of the disease is recent. HLB has been confirmed in only two adjacent commercial citrus groves of grapefruit and sweet orange. A study was conducted to compare the population of Candidatus Liberibacter asiaticus (CLas) cells in different plant parts including peduncle, columella, leaves, seeds, young shoots, flower buds, flowers, and bark of 6-year-old known infected grapefruit and sweet orange trees. The bacterial population was estimated using a previously described grand universal regression equation Y = 13.82 – 0.2866X, where Y is the log of the target copy number and X is the Ct (threshold cycle) of the assay. Except for bark tissue, there was no significant difference in the concentration of CLas cells in other plant parts between the two cultivars. Within the cultivar, the bacterial concentration also varied with the plant part, with peduncle, columella, midrib having significantly higher titer of CLas compared with other plant parts. The obtained results here are in agreement with previous studies conducted on Florida samples, but the consistently lowest bacterial titer recorded in young shoots, leaf blade, and especially leaf margins relative to the midrib has never been previously reported.

Accurate quantitative assessment of greasy spot of grapefruit, caused by Mycosphaerella citri Whiteside, is crucial for designing appropriate disease management strategies and estimating yield losses. A visual rating scale was developed by measuring the total leaf area of 143 ‘Rio Red’ grapefruit leaf samples, and calculating the percent diseased area. The mean severity of the entire sample was 23.1%, but many leaves on the orchard floor exhibited symptoms on more than 50.0% of the leaf area. This rating scale should be highly useful to growers and researchers for field assessments of greasy spot severity.

The Asian citrus psyllid preferentially feeds and exclusively reproduces on young, newly emerged flush shoots of citrus. Asian citrus psyllid nymphs feed and complete their life stages on these flush shoots. Recent studies conducted under greenhouse conditions have shown that the transmission rates of ‘Candidatus Liberibacter asiaticus’ (CLas), the putative causal agent of huanglongbing disease of citrus, are enhanced when flush shoots are present. However, it is unclear if CLas acquisition by migrant adult Asian citrus psyllids is similarly enhanced. To address this knowledge gap, cohorts of Asian citrus psyllid adults were allowed 1-wk acquisition access period (AAP) on flushing and nonflushing shoots of qPCR-tested symptomatic (CLas+) and asymptomatic (CLas–) 10-yr-old sweet orange trees under field conditions. After the AAP, they were tested for CLas by qPCR. Progeny Asian citrus psyllid adults that emerged 4 wk post-AAP were similarly retrieved and tested. Eighty percent of flushing and 30% of nonflushing CLas+ trees produced infective Asian citrus psyllid adults, indicating that flush shoots have greater potential to be inoculum sources for CLas acquisition. Concomitantly, 21.1% and 6.0% infective adults were retrieved, respectively, from flushing and nonflushing CLas+ trees, indicating that Asian citrus psyllid adults acquire CLas more efficiently from flush shoots relative to mature shoots. In addition, 12.1% of infective Asian citrus psyllid adult progeny were obtained from 70% of flushing CLas+ trees. Significantly lower mean Ct values were also obtained from infective adults retrieved from flushing relative to nonflushing trees. The results underscore the role of flush shoots in CLas acquisition and the need to protect citrus trees from Asian citrus psyllid infestations during flush cycles.

The study was conducted to investigate the optimal hormone treatment for rooting in bitter melon and the effect of defoliation on rooting and polyamine levels. Commercial preparation (diluted 1:10 and 1:20) gave extensive rooting within five days after treatment. The presence of leaf with the stem cutting was necessary for optimal rooting as defoliation drastically reduced rooting even when the rooting time was extended from 5 to 10 days. The presence of leaf also promoted the levels of putrescine three days after the hormone treatment just at the time of root emergence. Cuttings that did not have leaf did not show an increase in putrescine levels and did not produce roots. Spermidine levels also increased in the cuttings that rooted but there was no change in the levels of spermine in the cuttings that rooted. It appears that putrescine and spermidine play a role in regulating rooting in bitter melon cuttings. The vegetative propagation conditions described show that it is a practically viable technique for small growers to produce large number of plantlets at the farm for selection and cultivation.

The article “A new diagnostic real-time PCR method for huanglongbing detection in citrus root tissue”, written by Jong Won Park, Eliezer S. Louzada, W. Evan Braswell, Philip A. Stansly, John V. da Graca, Greg McCollum, John E. Rascoe, and Madhurababu Kunta, was originally published electronically on the publisher’s internet portal (currently SpringerLink) on 13 June 2018 without open access.

Citrus viroid-induced resistance to Phytophthora infection in citrus was measured by the number of Phytophthora sporangia in ‘Rio Red’ grapefruit (Citrus paradisi Macf.) bait tissue infected with citrus viroids compared with non-inoculated controls. Different viroid isolates containing mixtures of viroids [Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd), Citrus viroid III (CVd-III), Citrus viroid IV (CVd-IV)] were designated by plant numbers and sources. Source 13E was associated with the lowest number of sporangia in bark, leaves, and roots used as baits, whereas CEVd E9, a known severe CEVd isolate, significantly reduced the number of sporangia in leaves and bark. Sources 1A, 2E, 3E, 4D, and 6E showed a significantly reduced number of sporangia on bark, leaves, and roots compared with healthy plants and 44A; however, their effect was not as pronounced as that of E9 and 13E. Sources 12E and 44A did not suppress sporangia production. Previous reverse transcriptase–polymerase chain reaction analysis showed that all source plants had mixed infections with several viroids, whereas 12E and 44A contained no viroids. In addition to confirming the earlier reports on the suppression of Phytophthora infection in general, our study showed significantly reduced Phytophthora sporangia development resulting from a number of viroids in mixed infection, but there did not appear to be any effect related to viroid species. To determine if concentration affected resistance to Phytophthora, phenolic acids were extracted. Extraction of phenolic acids with 80% ethanol was more efficient compared with 100% methanol and an acetonitrile–water mixture. High-performance liquid chromatography revealed no notable detection of salicylic acid in healthy and viroid-infected plants, but there was a small peak corresponding to salicylic acid in Phytophthora-infected and both viroid and Phytophthora-infected plants. Flavone was detected in all the source plants with a slight increase in Phytophthora-infected and both viroid and Phytophthora-infected plants. A peak corresponding to quercetin dehydrate was detected in Phytophthora-infected plants. Efficient use of the right viroid isolate(s) can result in suppression of Phytophthora infection of citrus.

Ascorbate peroxidase is the key enzyme in detoxifying hydrogen peroxide (H2O2) and other reactive oxygen species from chloroplast and cytosol. A cDNA encoding a putative citrus ascorbate peroxidase, APXcit was isolated from mature ‘Dancy’ tangerine (Citrus reticulata Blanco) juice vesicles using differential display reverse transcription-polymerase chain reaction. The full-length APXcit sequence was composed of 1,082 bp nucleotides, including an open reading frame of 753 bp, putatively encoding a protein of 250 amino acids with a predicted molecular mass of 27 kDa. The 5′ untranslated region consisted of 90 nucleotides and the 3′ untranslated region of 239. The genomic clone of 2,457 bp was composed of 8 introns and 9 exons. A homology search for APXcit at the GenBank database showed high similarity to ascorbate peroxidase from several plant species. Southern analysis revealed that there may be 2–4 APXcit gene copies in citrus.

Colletotrichum fungi cause anthracnose and post-harvest rots on a wide range of plant species resulting in serious economic losses. In April 2017, leaf samples showing typical anthracnose symptoms of circular to oval brown spots and shot hole appearance in mature leaves where center of leaf spot fell out were collected from a Persian lime tree (Citrus x latifolia). The tree is located in a residential property in Corpus Christi, TX. Four tiny pieces (1-2 mm) of infected tissue from the lesions were surface sterilized for 2 min in 70% ethanol followed by 1 min in 3% sodium hypochlorite, plated on potato dextrose agar (Sigma-Aldrich), and incubated at 24 ±1°C. The four isolates obtained showed grey mycelium, numerous acervuli with dark bases, and orange conidial ooze. The conidia were hyaline, cylindrical with rounded ends, occasionally slightly constricted near center and appressoria were globose. Perithecia were not seen on either leaves or in cultures. DNA sequences were generated from seven loci known t...

Viroids are graft- or mechanically transmissible agents, disseminated through budding. Biological indexing of commercially important citrus cultivars grown in the Lower Rio Grande Valley of Texas showed that many are infected with citrus viroids. Most of these trees carried more than one viroid. In most cases, the infected trees are asymptomatic carriers because sour orange, the predominant rootstock used in Texas, does not show symptoms of viroid infection. Detection of viroids through biological indexing on sensitive indicator plants followed by sequential polyacrylamide gel electrophoresis (sPAGE) is the gold standard but is time-consuming and requires plants to be kept at optimum conditions. A conditional use of reverse transcriptase–polymerase chain reaction (RT-PCR) provides an efficient and alternative detection of viroids for use in the Texas virus-free citrus budwood certification program. RT-PCR could be useful in Texas to help expedite the evaluation for the presence of viroids before conducting the final biologic indexing. Using RT-PCR, we could detect, clone, and sequence full-length viroids of Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd) (both cachexia and noncachexia variants), Citrus viroid-III (Citrus dwarfing viroid), and Citrus viroid-IV (Citrus bark cracking viroid) from a collection of viroid-inoculated grapefruit plants. The source plants were previously shown to be viroid-infected by biological indexing on Etrog citron plants. Based on our results, RT-PCR can be a conditional substitute for biological indexing of mother trees in foundation blocks and shoot tip-grafted trees in the virus-free budwood program. A positive RT-PCR result has a serendipitous value because those trees can be discarded from the pool before expensive biological indexing.

A grapefruit tree on sour orange rootstock in a residential property in Mission, TX was suspected to have citrus dry root rot disease based on symptoms. The causal organism was isolated from the infected root samples and based on fungal cultural and microscopic morphology and PCR, it was confirmed to be Fusarium solani (Martius) Appel & Wollenweber emend. Snyder & Hansen. A total of 10 healthy sour orange rootstock seedlings were inoculated using conidial suspension of the fungus by the standard root-dip method. After 9 month post inoculation, the inoculated fungus was re-isolated from root and stem sections of these plants. Plants were smaller in size and displayed the classical symptoms of dry rot. The fungal colonies were confirmed to be F. solani based on fungal morphology and PCR. This is the first report of F. solani infecting sour orange rootstock plants in Texas.

The Ca 2+ -dependent protein phosphatase calcineurin is an important regulator of ion transporters from many organisms, including the Saccharomyces cerevisiae vacuolar Ca 2+ /H + exchanger Vcx1p. In yeast and plants, cation/H + exchangers are important in shaping cytosolic Ca2 + levels involved in signal transduction and providing tolerance to potentially toxic concentrations of cations such as Ca 2+ , Mn 2+ and Cd 2+ . Previous genetic evidence suggested Vcx1p is negatively regulated by calcineurin. By utilizing direct transport measurements into vacuolar membrane vesicles, we demonstrate that Vcx1p is a low-affinity Ca 2+ transporter and may also function in Cd 2+ transport, but cannot transport Mn 2+ . Furthermore, direct Ca 2+ transport by Vcx1p is calcineurin sensitive. Using a yeast growth assay, a mutant allele of VCX1 (VCX1-5204A/L208P), termed VCX1-M1, was previously found to confer strong Mn 2+ tolerance. Here we demonstrate that this Mn 2+ tolerance is independent of the Ca 2+ /Mn 2+ -ATPase Pmr1p and results from Mn 2+ -specific vacuolar transport activity of Vcx1-M1p. This Mn 2+ transport by Vcx1-M1p is calcineurin dependent, although the localization of Vcx1-M1p to the vacuole appears to be calcineurin independent. Additionally, we demonstrate that mutation of L208P alone is enough to confer calcineurin-dependent Mn 2+ tolerance. This study demonstrates that calcineurin can positively regulate the transport of cations by VCX1-M1p.

The presence of leaves in citrus inhibit axillary bud break which were released, in several citrus cultivars, as soon as leaves were removed. Since even the presence of small petiole alone was as effective in inhibiting bud growth in Satsuma trees as the whole leaf, this indicates that the supply of sugars or photosynthesis play little role in regulating bud break in Owari Satsuma trees. It appears that physical injury through complete defoliation may trigger hormonal stimulation that in turn initiates the metabolic processes leading to bud break. Majority of bud break from defoliation developed vegetative growth, but, as anticipated, chilling treatment in defoliated plants resulted in majority of buds breaking into flowers. Since differentiation of buds into vegetative or reproductive structures occur in the absence of leaves it indicates that in grapefruit, and possibly other citrus cultivars, metabolic processes leading to flowering occur within the resting bud tissue. Changes in the levels of specific polyphenols occur when buds begin to sprout as vegetative shoot, marked by increase in chlorogenic acid and naringenin levels. When buds develop into reproductive structures then such developmental change accompany dramatic rise in the levels of hesperidin, naringenin, and Apigenin-7-glucoside. In general, the results clearly show a strong inhibition of axillary bud sprouting even with the presence of a small portion of the leaf. Very distinct changes in the levels of specific polyphenols were associated with bud sprouting to vegetative shoot and when it develops into flower.

Huanglongbing (HLB), also known as citrus greening, is a destructive citrus disease associated with 3 α-proteobacteria species of Candidatus Liberibacter. The first report of HLB in the USA was from Florida in 2005 and Ca. L. asiaticus (Las) is the only species currently confirmed in the USA. In January 2012, a Valencia sweet orange tree in a commercial orchard in San Juan, Texas, tested positive for Las by real-time and conventional PCR assays and by the sequence of its partial 16S rRNA gene. The sample tested negative for Ca. L. americanus and Ca. L. africanus. All 4 Valencia sweet orange seedlings that were graft-inoculated using budwood from the first Texas HLB-infected tree showed typical HLB symptoms 3 months post-inoculation and tested positive for the pathogen. Such HLB typical symptoms as leaf blotchy mottle, twig die-back, veinal chlorosis, lopsided and greening fruits were observed on the Las-positive tree in the orchard, which immediately triggered an intensive survey of the disease in the area. Typical HLB symptoms were found on 54 Valencia sweet orange trees in the same orchard and 18 Rio Red grapefruit trees in an adjacent orchard. All these symptomatic trees tested positive for Las by PCR and sequencing.

Author(s): Kunta, Madhurababu; de La Garza, Carolina; da Graca, John V.; Setamou, Mamoudou; Louzada, Eliezer S. | Abstract: A total of 108 root and corresponding symptomatic leaf samples from four different quadrants were collected from 27 6 year old sweet orange trees in which the presence of Candidatus Liberibacter asiaticus (CLas) was previously confirmed by quantitative polymerase chain reaction (qPCR). There was no significant difference (pg0.05) in the level of infection as determined by threshold cycle (Ct) values between different types of tissue tested. Analysis of variance (ANOVA) showed that there is no significant difference (pg0.05) in test results among different distances from the trunk or quandrants where the root samples were collected. The stumps of the infected trees were covered in a psyllid-proof cage and leaves from the new shoots emerged from these stumps did not show the presence of CLas where as the roots showed the presence of CLas.n Moreover, there was no significant difference between roots from infected trees and roots from infected stumps.n Additionally, there was a significant difference with an average Ct value difference of 2.97 cycles between the DNA samples extracted from roots using two different commercially available kits.

Author(s): Setamou, M.; da Graca, J. V.; Kunta, M. | Abstract: After six years of intensive statewide surveys, Huanglongbing (HLB) was first detected in two adjacent commercial groves in Texas in 2012, and Candidatus Liberibacter asiaticus (CLas) was found associated with the disease. Assessment of affected trees within the two infected groves was made by visual inspection of foliage and PCR-testing of symptomatic tissue. HLB-infected trees exhibited an aggregated distribution and a strong perimeter effect in the two groves. While in one grove (sweet orange), more qPCR positive trees were observed in the south-eastern side of the grove, in the other (grapefruit) more HLB-infected were found on the western border, adjacent to the sweet orange grove suggesting movement of infected psyllids between the two groves. The Asian psyllid vector, Diaphorina citri Kuwayama, known vector of CLas, reported for the first time in 2001, is widespread in Texas. In an effort to reduce the risk of HLB in Texas, a proactive area-wide psyllid control program was initiated in 2010 which has contributed to significant reduction of psyllid populations in Texas. The detection of HLB has led to an intensification of psyllid control measures in groves within a mile radius of the HLB-positive sites, both in commercial and residential settings. All known infected trees have been removed and a five mile-radius quarantine has been put in place to regulate movement of all Rutaceae plants and to ensure safe harvesting of citrus.

RNA sequencing (RNA-seq) methods provide a complete description of RNA transcripts including alternative splicing and small RNA characterization. RNA-seq performed in our laboratory on healthy and Huanglongbing (HLB)-infected young leaves revealed that 4,044 transcripts were up-regulated and 2,562 were down-regulated in the diseased trees. Moreover, a number of genes showed alternative splicing events including exon skipping, intron retention, and 5' and 3' alternative splicing. Furthermore, quantitative PCR (qPCR) performed on 20 randomly chosen genes with high differential expression (10 up and 10 down regulated) showed that all were consistent with RNA-seq data. Additionally, variation in levels of gene expression was observed between young and mature leaves. These early host plant response genes due to HLB-infection might be useful in the development of early HLB-detection methods before manifestation of disease symptoms in the infected plants.

Now that the presence of Huanglongbing (HLB) has been confirmed in California, protecting the state’s citrus industry through early detection of disease is essential in curtailing its spread. Because ‘Candidatus Liberibacter asiaticus’ (Las), the putative causal agent of HLB, accumulates in its vector, the Asian citrus psyllid (ACP), Diaphorina citri, Kuwayama, accurate testing of the insect is vital. Due to the fact that insect secondary metabolites interfere with downstream applications (1, 3) there is concern about the number of insects pooled in DNA extractions without compromising Las detection. The current USDA CPHST approved method of testing (2) limits the pooling size to five individual insects per extraction followed by the highly sensitive quantitative polymerase chain reaction (QPCR) detection technique. The QPCR reaction targets a region of the Las 16S ribosomal gene (4) and simultaneously the D. citri specific wingless (WGLS) gene (5) as an internal control for extraction efficiency. In contrast, the CDFA laboratory routinely pools up to 25 individual insects for DNA extraction, followed by the same QPCR detection. If pooling 25 individuals is indeed a safe practice all the labs currently limiting sample pooling to five individuals could save a substantial amount of time and money. We decided to approximate the pooling limit using single insect equivalents of DNA extracted from Las infected ACP from colonies at USDA-ARS in Fort Pierce, Florida pooled with intact ACP from clean UC Riverside, California, quarantine colonies. Depending on results we would follow these experiments with experiments using intact Las positive ACP.