In-Grove Spatiotemporal Spread of Citrus Huanglongbing and Its Psyllid Vector in Relation to Weather.

Reports of spatial patterns of 'Candidatus Liberibacter asiaticus'-infected asymptomatic citrus trees and 'Ca. L. asiaticus'-positive Asian citrus psyllids (ACP) are rare, as are published relationships between huanglongbing (HLB), ACP, and weather. Here, spatial patterns of 'Ca. L. asiaticus'-positive asymptomatic and symptomatic trees were determined every half year in a small grove over 2.5 years, and of HLB-symptomatic trees and ('Ca. L. asiaticus'-positive) ACP populations every month in two commercial groves for 1 year. Spread of symptomatic trees followed that of asymptomatic 'Ca. L. asiaticus'-positive trees with <6 months' delay. 'Ca. L. asiaticus'-positive asymptomatic and symptomatic fronts moved at 2.5 to 3.6 m month-1. No spatial relationship was detected between ACP populations and HLB-infected trees. HLB incidence and 'Ca. L. asiaticus'-positive ACP dynamics were tentatively positively correlated with monthly rainfall data and, to a lesser extent, with average minimum temperature.

Regional Spatial-Temporal Spread of Citrus Huanglongbing Is Affected by Rain in Florida.

Citrus huanglongbing (HLB), associated with 'Candidatus Liberibacter asiaticus' (Las), disseminated by Asian citrus psyllid (ACP), has devastated citrus in Florida since 2005. Data on HLB occurrence were stored in databases (2005 to 2012). Cumulative HLB-positive citrus blocks were subjected to kernel density analysis and kriging. Relative disease incidence per county was calculated by dividing HLB numbers by relative tree numbers and maximum incidence. Spatiotemporal HLB distributions were correlated with weather. Relative HLB incidence correlated positively with rainfall. The focus expansion rate was 1626 m month-1, similar to that in Brazil. Relative HLB incidence in counties with primarily large groves increased at a lower rate (0.24 year-1) than in counties with smaller groves in hotspot areas (0.67 year-1), confirming reports that large-scale HLB management may slow epidemic progress.

First Report of a 16SrIV-D Phytoplasma Associated with Texas Phoenix Palm Decline on Pigmy Date Palm (Phoenix roebelenii) in Florida.

The pigmy date palm (Phoenix roebelenii O'Brien) is used as an ornamental in Florida and is popular and lucrative within the nursery trade. Severe decline of several pigmy date palms was observed at a residence in Hillsborough County, FL. Several other palm species, including P. canariensis (Canary Island date palm), P. sylvestris (wild date palm), P. dactylifera (date palm), Syagrus romanzoffiana (queen palm), and Sabal palmetto (cabbage palm), in Florida are known to be affected by Texas Phoenix palm decline (TPPD), a disease associated with a phytoplasma subgroup strain 16SrIV-D (2,3). Moreover, the location of the affected pigmy date palms was in the proximity of many other diseased cabbage palms that were identified in previous surveys and subsequently rogued. Genomic DNA was extracted from 100 mg of ground-up palm trunk tissues containing phloem cells with a DNeasy Plant Mini kit column (QIAGEN Inc., Valencia, CA) from four specimens. A high-fidelity PCR (Hf-PCR) procedure was used in preference to standard PCR because it was 10,000 to 100,000 times more sensitive (1,4). The Hf-PCR (50 µl) utilized two DNA polymerases; Taq (five units) and ACCUZYME (one unit), 350 µM dNTP, a buffer (50 mM Tris pH 9.2, 16 mM ammonium sulfate, and 1.75 mM magnesium chloride), a higher concentration of primers (200 pM) (2,3), and palm DNA templates (>10 ng) or no DNA negative control. Hf-PCR was performed using three linked profiles: (i) 94°C (2 min) (1 cycle); (ii) 94°C (10 s), 50°C (30 s) for P1m/P7 or 57°C for LY16Sf/LY16Sr and 68°C (2 min) (10 cycles); and (iii) 94°C (10 s), 50°C (30 s) for P1m/P7 or 57°C for LY16Sf/LY16Sr and 68°C (2 min plus 20 s added for every consecutive cycle) (20 cycles) (1). The genomic DNA extracted from P. roebelenii specimens was used as template for amplification by Hf-PCR. Expected 1.8- and 1.4-kb DNA bands for each primer combination were readily amplified. The Hf-PCR products were sequenced (GenBank Accession No. JF791816) and a BLAST search revealed a 100% similarity with a phytoplasma subgroup strain 16SrIV-D (EF042899 and AF434989), which is known to cause severe palm decline (TPPD) in other hosts (2,3). To our knowledge, this is the first report of TPPD from P. roebelenii, and therefore, expands the host range of this pathogen. In areas where TPPD is present, the landscape industry may need to identify alternative nonhost palm species or resistant varieties for disease management. References: (1) W. M. Barnes. Proc. Natl. Acad. Sci. USA 91:2216, 1994. (2) N. A. Harrison et al. Plant Dis. 86:676, 2002. (3) N. A. Harrison et al. Ann. Appl. Biol. 153:85, 2008. (4) A. Jeyaprakash and M. A. Hoy. Insect Mol. Biol. 9:393, 2000.

Detection of 'Candidatus Liberibacter asiaticus' in Diaphorina citri and its importance in the management of citrus huanglongbing in Florida.

Citrus huanglongbing (HLB or citrus greening), is a highly destructive disease that has been spreading in both Florida and Brazil. Its psyllid vector, Diaphorina citri Kuwayama, has spread to Texas and Mexico, thus threatening the future of citrus production elsewhere in mainland North America. Even though sensitive diagnostic methods have been developed for detection of the causal organisms, Candidatus Liberibacter spp., the pathogen cannot be detected consistently in plants until symptoms develop, presumably because of low titer and uneven distribution of the causal bacteria in nonsymptomatic tissues. In the present study, TaqMan based real-time quantitative polymerase chain reaction methodology was developed for detection of 'Ca. L. asiaticus' in D. citri. Over 1,200 samples of psyllid adults and nymphs, collected from various locations in Florida, from visually healthy and HLB symptomatic trees at different times of the year were analyzed to monitor the incidence and spread of HLB. The results showed that spread of 'Ca. L. asiaticus' in an area may be detected one to several years before the development of HLB symptoms in plants. The study suggests that discount garden centers and retail nurseries may have played a significant role in the widespread distribution of psyllids and plants carrying HLB pathogens in Florida.

Biology and biotype determination of greenbug, Schizaphis graminum (Hemiptera: Aphididae), on seashore paspalum turfgrass (Paspalum vaginatum).

Greenbug, Schizaphis graminum (Rondani) (Hemiptera: Aphididae), was first discovered damaging seashore paspalum (Paspalum vaginatum Swartz) turfgrass in November 2003 at Belle Glade, FL. Inquiries to several golf courses with seashore paspalum turf across southern Florida indicated infestation was wide spread by April 2004. Damage symptoms progress from water soaked lesions surrounding feeding sites within 24 h to chlorosis and necrosis of leaf tips within 96 h. Problems caused by greenbug feeding were initially misdiagnosed as fertilizer, disease, other insects, or water management problems because aphids were not previously found on warm season turfgrasses. Greenbug development and fecundity studies were conducted on six seashore paspalum varieties: 'Aloha,' 'SeaDwarf,' 'SeaGreen,' 'SeaIsle,' 'SeaWay,' and 'SeaWolf.' Greenbug did not survive on 'SeaWolf.' Development rates (mean +/- SEM) ranged from 7.6 +/- 0.2 to 8.2 +/- 0.2 d on the remaining varieties. Greenbug longevity and fecundity on 'Aloha' were significantly less than on the other varieties. The estimated intrinsic rate of natural increase (r(m)) for greenbug ranged from 0.24 to 0.26 across tested varieties. Values for net reproductive rate (R(o)) ranged from 12.3 on 'Aloha' to 40.4 on 'SeaWay.' In feeding trials on indicator plants, the Florida isolate of greenbug exhibited a unique biotypic profile most commonly found on noncultivated grass hosts. It was virulent on the wheat variety GRS1201 that is resistant to the principal agricultural biotypes attacking small grains and to all currently available resistant sorghum varieties.

First Report of Dodder Transmission of Huanglongbing from Naturally Infected Murraya paniculata to Citrus.

Huanglongbing (HLB) or "greening" disease of citrus is caused by phloem-limited, uncultured bacteria in the genus "Candidatus Liberibacter". HLB is one of the most destructive diseases of citrus worldwide and is considered so dangerous to a U.S. citrus production that the USDA has listed "Ca. Liberibacter species" as a Select Agent. HLB is spread by the Asian citrus psyllid, Diaphorina citri, which was intercepted 40 times by APHIS/PPQ at U.S. ports between 1985 and 1998, became established in Florida by 1998, and more recently in Texas (1). HLB was first detected in the United States near Miami, FL during August 2005, and to date has been confirmed to have spread to 12 Florida counties. In addition to citrus, Murraya paniculata (orange jasmine) is a preferred host of D. citri, and retail trade in this ornamental shrub is strongly implicated in the distribution of D. citri (1). M. paniculata is reported to be a cryptic or largely asymptomatic host of "Ca. Liberibacter" (4), but another report concludes that the bacteria cannot replicate in M. paniculata (2). The epidemiological significance of murraya as a host for the HLB pathogen is therefore unclear. We report here the transmission of "Ca. Liberibacter asiaticus" from M. paniculata to citrus. Two M. paniculata plants, suspected of harboring "Ca. Liberibacter" because of their proximity to HLB-infected citrus and infested with D. citri, were removed from the field, treated with insecticide, and transferred to a quarantine facility. Both plants tested positive for "Ca. Liberibacter" by nested PCR using primers OI1 and OI2 (3) as the first set and primers CGO3F (RGG GAA AGA TTT TAT TGG AG) and CGO5R (GAA AAT AYC ATC TCT GAT ATC GT) as the second set. Two, young, sweet orange plants (Citrus sinensis) grown and maintained in psyllid-free greenhouses in Gainesville, FL were infected by dodder (Cuscuta pentagona) grown from seed. After the dodder had become well established on the orange plants, the orange plants were moved adjacent to the two murraya plants and the dodder from the citrus was draped over the murraya. Coinfection of murraya by dodder occurred within a few days. Sixty days later, both murraya plants, both sweet orange plants, and the connecting dodder all repeatedly tested positive for "Ca. Liberibacter" by nested PCR. Beginning 2 weeks later, the orange plants tested positive by standard PCR using primer set OI1 and OI2 or CGO3F and CGO5R, but remained without typical greening symptoms. Sequencing of the PCR products confirmed amplification of "Ca. L. asiaticus" DNA. We conclude that M. paniculata can serve as an infection source of a Select Agent since it can host the HLB pathogen for at least 2 months and the HLB pathogen can be transmitted to sweet orange during this time. References: (1) S. E. Halbert and K. L. Manjunath. Florida Entomol. 87:330, 2004. (2) T. H. Hung et al. J. Phytopathol. 148:321, 2000. (3) S. Jagoueix et al. Mol. Cell Probes 10:43, 1996. (4) T. Li and C. Ke. Acta Phytophylacica Sin. 29:31, 2002.

Seasonal abundance of aphids (Homoptera: Aphididae) in wheat and their role as barley yellow dwarf virus vectors in the South Carolina coastal plain.

Aphid (Homoptera: Aphididae) seasonal flight activity and abundance in wheat, Triticum aestivum L., and the significance of aphid species as vectors of barley yellow dwarf virus were studied over a nine-year period in the South Carolina coastal plain. Four aphid species colonized wheat in a consistent seasonal pattern. Greenbug, Schizaphis graminum (Rondani), and rice root aphid, Rhopalosiphum rufiabdominalis (Sasaki), colonized seedlingwheat immediately after crop emergence, with apterous colonies usually peaking in December or January and then declining for the remainder of the season. These two aphid species are unlikely to cause economic loss on wheat in South Carolina, thus crop managers should not have to sample for the subterranean R. rufiabdominalis colonies. Bird cherry-oat aphid, Rhopalosiphum padi (L.), was the second most abundant species and the most economically important. Rhopalosiphum padi colonies usually remained below 10/row-meter until peaking in February or March. Barley yellow dwarf incidence and wheat yield loss were significantly correlated with R. padi peak abundance and aphid-day accumulation on the crop. Based on transmission assays, R. padi was primarily responsible for vectoring the predominant virus serotype (PAV) we found in wheat. Pest management efforts should focus on sampling for and suppressing this aphid species. December planting reduced aphid-day accumulation and barley yellow dwarf incidence, but delayed planting is not a practical management option. English grain aphid, Sitobion avenae (F.), was the last species to colonize wheat each season, and the most abundant. Sitobion avenae was responsible for late-season virus transmission and caused direct yield loss by feeding on heads and flag leaves during an outbreak year.

Somaclonal variation in the progeny of transgenic barley.

Somaclonal variation (SCV) in transgenic plants may slow the incorporation of introduced genes into commercially competitive cultivars. Somaclonal variation in transgenic barley (Hordeum vulgare L.) was assessed in one experiment by comparing the agronomic characteristics of 44 segregating transgenic lines in the T2 generation to their non-transformed parent ('Golden Promise'). A second experiment examined the agronomic characteristics of seven transgenic-derived, null (non-transgenic) segregant lines in the T2 and T4 generations. Compared to their uncultured parent, Golden Promise, most of these lines were shorter, lower yielding, and had smaller seed, and the variability among individual plants was higher. The frequency and severity of the observed SCV was unexpectedly high, and the transformation procedure appeared to induce greater SCV than tissue culture in the absence of transformation. Attempts to understand the sources of SCV, and to modify transformation procedures to reduce the generation of SCV, should be made.