Tim R. Gottwald

Current Epidemiological Understanding of Citrus Huanglongbing

Huanglongbing (HLB) is the most destructive citrus pathosystem worldwide. Previously known primarily from Asia and Africa, it was introduced into the Western Hemisphere in 2004. All infected commercial citrus industries continue to decline owing to inadequate current control methods. HLB increase and regional spatial spread, related to vector populations, are rapid compared with other arboreal pathosystems. Disease dynamics result from multiple simultaneous spatial processes, suggesting that psyllid vector transmission is a continuum from local area to very long distance. Evolutionarily, HLB appears to have originated as an insect endosymbiont that has moved into plants. Lack of exposure of citrus to the pathogen prior to approximately 100 years ago did not provide sufficient time for development of resistance. A prolonged incubation period and regional dispersal make eradication nonviable. Multiple asymptomatic infections per symptomatic tree, incomplete systemic distribution within trees, and prolonged incubation period make detection difficult and greatly complicate disease control.

Xanthomonas axonopodis pv. citri: factors affecting successful eradication of citrus canker

UNLABELLED SUMMARY Taxonomic status: Bacteria, Proteobacteria, gamma subdivision, Xanthomodales, Xanthomonas group, axonopodis DNA homology group, X. axonopodis pv. citri (Hasse) Vauterin et al. Microbiological properties: Gram negative, slender, rod-shaped, aerobic, motile by a single polar flagellum, produces slow growing, non-mucoid colonies in culture, ecologically obligate plant parasite. HOST RANGE Causal agent of Asiatic citrus canker on most Citrus spp. and close relatives of Citrus in the family Rutaceae. Disease symptoms: Distinctively raised, necrotic lesions on fruits, stems and leaves. EPIDEMIOLOGY Bacteria exude from lesions during wet weather and are disseminated by splash dispersal at short range, windblown rain at medium to long range and human assisted movement at all ranges. Crop loss: Severe infections cause defoliation, blemished fruit, premature fruit drop, die-back of twigs and general debilitation of the tree. Distribution: Citrus canker is not present in all subtropical to tropical regions of citriculture in the world, so considerable regulatory efforts are expended to prevent the introduction and spread of X. axonopodis pv. citri into areas in the Americas, Australia and elsewhere, with climates conducive to the disease. IMPORTANCE Limited strategies exist for suppression of citrus canker on more susceptible cultivars. Blemished fruit are unmarketable and exposed fruit are restricted in market access. The economic impact of loss of markets is much greater than that from yield and quality reductions of the crop. USEFUL WEBSITES http://doacs.state.fl.us/canker, http://www.apsnet.org/education/lessonsplantpath/citruscanker/top.htm, http://www.apsnet.org/online/feature/citruscanker/, http://www.plantmanagementnetwork.org/pub/php/review/citruscanker/, http://www.abecitrus.com.br/fundecitrus.html, http://www.biotech.ufl.edu/PlantContainment/canker.htm, http://www.aphis.usda.gov/oa/ccanker/.

Plant Disease Severity Estimated Visually, by Digital Photography and Image Analysis, and by Hyperspectral Imaging

Reliable, precise and accurate estimates of disease severity are important for predicting yield loss, monitoring and forecasting epidemics, for assessing crop germplasm for disease resistance, and for understanding fundamental biological processes including co-evolution. Disease assessments that are inaccurate and/or imprecise might lead to faulty conclusions being drawn from the data, which in turn can lead to incorrect actions being taken in disease management decisions. Plant disease can be quantified in several different ways. This review considers plant disease severity assessment at the scale of individual plant parts or plants, and describes our current understanding of the sources and causes of assessment error, a better understanding of which is required before improvements can be targeted. The review also considers how these can be identified using various statistical tools. Indeed, great strides have been made in the last thirty years in identifying the sources of assessment error inherent to visual rating, and this review highlights ways that assessment errors can be reduced—particularly by training raters or using assessment aids. Lesion number in relation to area infected is known to influence accuracy and precision of visual estimates—the greater the number of lesions for a given area infected results in more overestimation. Furthermore, there is a widespread tendency to overestimate disease severity at low severities (<10%). Both interrater and intrarater reliability can be variable, particularly if training or rating aids are not used. During the last eighty years acceptable accuracy and precision of visual disease assessments have often been achieved using disease scales, particularly because of the time they allegedly save, and the ease with which they can be learned, but recent work suggests there can be some disadvantages to their use. This review considers new technologies that offer opportunity to assess disease with greater objectivity (reliability, precision, and accuracy). One of these, visible light photography and digital image analysis has been increasingly used over the last thirty years, as software has become more sophisticated and user-friendly. Indeed, some studies have produced very accurate estimates of disease using image analysis. In contrast, hyperspectral imagery is relatively recent and has not been widely applied in plant pathology. Nonetheless, it offers interesting and potentially discerning opportunities to assess disease. As plant disease assessment becomes better understood, it is against the backdrop of concepts of reliability, precision and accuracy (and agreement) in plant pathology and measurement science. This review briefly describes these concepts in relation to plant disease assessment. Various advantages and disadvantages of the different approaches to disease assessment are described. For each assessment method some future research priorities are identified that would be of value in better understanding the theory of disease assessment, as it applies to improving and fully realizing the potential of image analysis and hyperspectral imagery.

The Citrus Canker Epidemic in Florida: The Scientific Basis of Regulatory Eradication Policy for an Invasive Species

Increasing international travel and trade have rendered U.S. borders much more porous and dramatically increased the risk of introductions of invasive plant pests into agricultural crops (1). Nationally, the responsibility for safeguarding agriculture falls to the U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Plant Protection and Quarantine (USDA, APHIS, PPQ). However, the current system for protecting agricultural industries has broken down allowing for an unprecedented number of introductions of exotic pests, including plant pathogens. Such introductions threaten crops and can hinder national and international agricultural markets and trade. In 1999, President Clinton announced an initiative for invasive species to address these issues and introduced an Executive Order on Invasive Species (10). The order is intended to coordinate and enhance federal government efforts to prevent introduction of invasive species and to provide for their control. The Executive Order calls for the appointment of a council, chaired by the Secretaries of Agriculture, Commerce, and the Interior, and composed of key federal agencies charged with developing an invasive species management plan. In response, the National Plant Board initiated a “stakeholder” review of the USDA, APHIS, PPQ safeguarding system in order to identify means to improve pest exclusion and detection of and response to invasive pest introductions (1). Enter citrus canker. Currently in Florida, one such invasive species is Xanthomonas axonopodis pv. citri (Xac), a bacterial plant pathogen that causes Asiatic citrus canker. Citrus canker is an introduced plant disease that has received considerable press attention, has produced far-reaching political and socioeconomic impact in Florida, and has implications for national and international trade (3,4). Although Xac is mostly a leaf and fruit-blemishing pathogen, the disease triggers immediate quarantines of areas with outbreaks in Florida, disrupting movement of fresh fruit (3,11). The importance of citrus canker as a major invasive disease and the political forces that govern attempts to eradicate the disease were recently exemplified by the visit of the U.S. Secretary of Agriculture to the Homestead/Florida City area south of Miami on 22 March 2000 to view a major outbreak of the disease first hand. Secretary Glickman declared the South Florida canker outbreak area in Dade and Broward Counties a federal disaster area, opening the door for disaster relief funds and for U.S. Marshals to assist crews in expediting the cutting and removal of trees from homeowners’ residences in metropolitan Miami (26). South Florida was one stop on a national tour conducted by the Secretary of Agriculture to highlight non-native, invasive pests and diseases that threaten U.S. agriculture and natural resources (20). Citrus canker has a long history. The disease was first found spread throughout the southeastern United States on imported seedlings from Japan in the early 1910s and was declared eradicated from Florida and the adjacent states in 1933. Citrus canker was discovered again in Manatee County Florida, south of Tampa Bay in the late 1980s, and was thought to be eradicated by 1994. Two years thereafter, the disease re-emerged in the same area on the west coast of Florida where the 1980s outbreak had occurred. In the meantime, a new and separate infestation of citrus canker was discovered in urban Miami in 1995, that was estimated to have been introduced in 1992 or 1993 (17). Since that time the disease has spread from an initial area of 14 square miles near the Miami airport, to over 1,005 square miles in the metropolitan area plus an additional 260 square miles of urban and commercial citrus areas throughout the State. Genomic analysis of bacterial isolates from both time periods indicates that the latest Manatee County outbreak is a hold over from the 1980s outbreak that escaped the eradication program. However, the majority of current outbreaks of citrus canker are caused by the same genotype of Xac as in the Miami area. Thus, human-assisted movement of the bacterium from that source appears to have occurred several times. In early 2000, a third genomically distinct isolate of Asiatic citrus canker with an attenuated host range was identified in Palm Beach County on the east coast of Florida. Thus, there are presently at least three Xac genotypes that have been introduced into Florida in the recent two decades. Although this bacterial disease is mostly a leaf and fruit-spotting malady, it results in immediate quarantines, disrupting national and international trade (3,11). Although citrus canker can cause debilitation of trees and losses in fruit quality and yield, it is because of its socioeconomic and political impact that the disease is so devastating. If Xac should become endemic in Florida, it will effectively result in a prohibition of interstate commerce of fresh citrus fruit, which comprises approximately 20% of the State’s

Relationships Between Disease Incidence at Two Levels in a Spatial Hierarchy

8 billion commercial citrus industry (23). In addition, some cultivars of highly susceptible citrus species, such as grapefruit (Citrus paradisi), will have reduced economic viability due to the requirement for multiple bactericidal sprays per year to maintain yields and quality. The Florida citrus industry is concentrated predominantly in the southern half of the State, in close proximity to rapidly expanding urban population centers. Because the outbreaks originated in urban areas, response to citrus canker, therefore, affects not just the citrus industry. Hundreds of thousands of urban homeowners have citrus trees as ornamentals and for dooryard fruit production and have had or will have their trees destroyed. The Florida Department of Agriculture and Consumer Services, Division of Plant Industry (DPI) and USDA, APHIS responded to the 1995 Corresponding author: T. R. Gottwald; E-mail address: TGottwald@ushrl.ars.usda.gov

Spatial and Temporal Analyses of Citrus Sudden Death as a Tool to Generate Hypotheses Concerning Its Etiology

ABSTRACT Relationships between disease incidence measured at two levels in a spatial hierarchy are derived. These relationships are based on the properties of the binomial distribution, the beta-binomial distribution, and an empirical power-law relationship that relates observed variance to theoretical binomial variance of disease incidence. Data sets for demonstrating and testing these relationships are based on observations of the incidence of grape downy mildew, citrus tristeza, and citrus scab. Disease incidence at the higher of the two scales is shown to be an asymptotic function of incidence at the lower scale, the degree of aggregation at that scale, and the size of the sampling unit. For a random pattern, the relationship between incidence measured at two spatial scales does not depend on any unknown parameters. In that case, an equation for estimating an approximate variance of disease incidence at the lower of the two scales from incidence measurements made at the higher scale is derived for use in the context of sampling. It is further shown that the effect of aggregation of incidence at the lower of the two scales is to reduce the rate of increase of disease incidence at the higher scale.

Detection and Characterization of a New Strain of Citrus Canker Bacteria from Key/Mexican Lime and Alemow in South Florida

ABSTRACT Citrus sudden death (CSD), a new disease of unknown etiology that affects sweet orange grafted on Rangpur lime, was visually monitored for 14 months in 41 groves in Brazil. Ordinary runs analysis of CSD-symptomatic trees indicated a departure from randomness of symptomatic trees status among immediately adjacent trees mainly within rows. The binomial index of dispersion (D) and the intraclass correlation (k) for various quadrat sizes suggested aggregation of CSD-symptomatic trees for almost all plots within the quadrat sizes tested. Estimated parameters of the binary form of Taylors power law provided an overall measure of aggregation of CSD-symptomatic trees for all quadrat sizes tested. Aggregation in each plot was dependent on disease incidence. Spatial autocorrelation analysis of proximity patterns suggested that aggregation often existed among quadrats of various sizes up to three lag distances; however, significant lag positions discontinuous from main proximity patterns were rare, indicating a lack of spatial association among discrete foci. Some asymmetry was also detected for some spatial autocorrelation proximity patterns, indicating that within-row versus across-row distributions are not necessarily equivalent. These results were interpreted to mean that the cause of the disease was most likely biotic and its dissemination was common within a local area of influence that extended to approximately six trees in all directions, including adjacent trees. Where asymmetry was indicated, this area of influence was somewhat elliptical. Longer-distance patterns were not detected within the confines of the plot sizes tested. Annual rates of CSD progress based on the Gompertz model ranged from 0.37 to 2.02. Numerous similarities were found between the spatial patterns of CSD and Citrus tristeza virus (CTV) described in the literature, both in the presence of the aphid vector, Toxoptera citricida. CSD differs from CTV in that symptoms occur in sweet orange grafted on Rangpur lime. Based on the symptoms of CSD and on its spatial and temporal patterns, our hypothesis is that CSD may be caused by a similar but undescribed pathogen such as a virus and probably vectored by insects such as aphids by similar spatial processes to those affecting CTV.

Differential host range reaction of citrus and citrus relatives to citrus canker and citrus bacterial spot determined by leaf mesophyll susceptibility

In the Wellington and Lake Worth areas of Palm Beach County, FL, citrus canker appeared on Key/Mexican lime (Citrus aurantiifolia) and alemow (C. macrophylla) trees over a period of about 6 to 7 years before detection, but nearby canker-susceptible citrus, such as grapefruit (C. × paradisi) and sweet orange (C. sinensis), were unaffected. Colonies of the causal bacterium, isolated from leaf, stem, and fruit lesions, appeared similar to the Asiatic group of strains of Xanthomonas axonopodis pv. citri (Xac-A) on the nutrient agar plate, but the growth on lima bean agar slants was less mucoid. The bacterium produced erumpent, pustule-like lesions of typical Asiatic citrus canker syndrome after inoculation into Key/Mexican lime, but brownish, flat, and necrotic lesions on the leaves of Duncan grapefruit, Madame Vinous sweet orange, sour orange (C. aurantium), citron (C. medica), Orlando tangelo (C. reticulata × C. × paradisi), and trifoliate orange (Poncirus trifoliata). The bacterium did not react with the Xac-A specific monoclonal antibody A1 using enzyme-linked immunosorbent assay (ELISA) and could not be detected by polymerase chain reaction (PCR)-based assays using primers selected for Xac-A. DNA reassociation analysis confirmed that the pathogen, designated as Xac-AW, was more closely related to Xac-A and Xac-A* strains than X. axonopodis pv. aurantifolii or the citrus bacterial spot pathogen (X. axonopodis pv. citrumelo). The strain can be easily differentiated from Xac-A and Xac-A* using ELISA, PCR-based tests, fatty acid analysis, pulsed-field gel electrophoresis of genomic DNA, and host specificity.

Environmental Factors Affecting Production, Release, and Field Populations of Conidia of Alternaria alternata, the Cause of Brown Spot of Citrus

The leaf mesophyll susceptibility of 54 citrus species, cultivars, and relatives to Xanthomonas campestris pv. citrumelo, the cause of citrus bacterial spot, was evaluated in Hastings, Florida, during 1989 and 1990. A similar host range of 53 citrus species, cultivars, and relatives was tested in Beltsville, Maryland, during 1991 to compare their differential susceptibility to X. c. citri, which causes citrus canker, and to X. c. citrumelo by inoculations on foliage of the same trees in replicated field plots. Field-grown trees were pruned to stimulate synchronous leaf flush for inoculation by a modified pinprick method. Lesion size at 60 days (Hastings plots) or 45 days (Beltsville plots) postinoculation was used to quantify leaf mesophyll susceptibility. For X. c. citrumelo inoculations, lesion expansion was greatest on cultivars of trifoliate orange and trifoliate orange hybrids. Smaller lesions formed on Citrus spp. such as grapefruit, sweet orange, sour orange, mandarin, lemon, and their hybrids, with the exception of Key lime, which developed lesions similar to those formed on trifoliate hybrids. Susceptibility of most citrus types to X. c. citri was more general. Lesion sizes resulting from pinprick inoculations with X. c. citri were not significantly different among Citrus spp. and hybrids, indicating a general susceptibility of leaf mesophyll. Smaller lesions generally formed on citrus relatives, including some cultivars of trifoliate orange. Because pinprick inoculations cause wounds and open the leaf mesophyll to direct colonization by bacteria, this method bypasses stomatal infection and does not consider other factors that may affect field resistance.

Spatial Pattern Analysis of Sharka Disease (Plum pox virus Strain M) in Peach Orchards of Southern France

ABSTRACT Alternaria brown spot, caused by Alternaria alternata pv. citri, affects many tangerines and their hybrids, causing loss of immature leaves and fruit and reducing the marketability of the remaining fruit. Conidial production of A. alternata was greatest on mature leaves moistened and maintained at near 100% relative humidity (RH) for 24 h, whereas leaves that had been soaked or maintained at moderate RH produced few conidia. Conidial release from filter paper cultures and infected leaves was studied in a computer-controlled environmental chamber. Release of large numbers of conidia was triggered from both substrates by sudden drops in RH or by simulated rainfall events. Vibration induced release of low numbers of conidia, but red/infrared irradiation had no effect. In field studies from 1994 to 1996, air sampling with a 7-day recording volumetric spore trap indicated that conidia were present throughout the year with periodic large peaks. The number of conidia captured was not closely related to rainfall amounts or average wind speed, but was weakly related to the duration of leaf wetness. Likewise, disease severity on trap plants placed in the field weekly during 1995 to 1996 was not closely related to conidial numbers or rainfall amounts, but was weakly related to leaf wetness duration. Sufficient inoculum appears to be available to allow infection to occur throughout the year whenever susceptible host tissue and moisture are available.