Journal of Citrus Pathology

The pathogens Candidatus Liberibacter asiaticus (Las) and Citrus tristeza virus (CTV) are both phloem limited and have significant economic impact on citrus production wherever they are found. Studies of host resistance have indicated that Poncirus trifoliata has tolerance or resistance to both pathogens, suggesting that there may be some common factors in the 2 kinds of resistance. We have conducted studies of host gene expression changes that occur in response to infection to gain further insight. Controlled inoculation by grafting infected budwood was used to infect potted greenhouse plants of Cleopatra mandarin (Citrus reticulata), US-897 (C. reticulata x P. trifoliata), and US-942 (C. reticulata x P. trifoliata) with CTV and with Las, the pathogen associated with the disease huanglongbing (HLB). Stem and leaf tissue was collected at 10, 20, and 30 weeks after inoculation, DNA and RNA were extracted and subjected to qPCR and RT-qPCR analysis. Few differences in gene expression were observed between mock-inoculated and CTV-inoculated plants. Differences between mock-inoculated and Las-inoculated plants were most pronounced in susceptible Cleopatra plants and at the later stages of infection. Notable was the higher expression of a gene for miraculin-like protein 2 and other defense-related genes in US-897 and US-942 plants independent of infection. It is hypothesized that tolerance or resistance of US-897 and US-942 is associated with a higher constitutive expression of defense-related or other genes associated with the P. trifoliata parentage, rather than with induced expression in response to bacterial infection.

In São Paulo State (SPS), sweet orange (Citrus sinensis) trees with huanglongbing (HLB) symptoms are infected with Candidatus (Ca.) Liberibacter (L.) asiaticus (Las) or Ca. L. americanus (Lam). However, in 2007, 3 years after HLB was first reported in SPS, some trees with characteristic HLB symptoms were found free of liberibacters, but infected with a phytoplasma of 16Sr group IX. This phytoplasma was further characterized by PCR amplification of ribosomal protein genes rpsC-rplV-rpsS and amplicon sequencing. A qPCR test to detect the phytoplasma in plants and insects was also developed on the basis of the ribosomal protein genes. The phytoplasma was transmitted from citrus-to-citrus by grafting. The 16Sr group IX phytoplasma associated with HLB symptoms in sweet orange in SPS and characterized by the above techniques was named “HLB-phytoplasma”. Although the HLB-phytoplasma is widely distributed in many municipalities of central, northern, and northwestern SPS, the number of HLB-phytoplasma-infected trees in each municipality is very small. Experiments have been undertaken to identify the origin of the HLB-phytoplasma and the source of inoculum on which a putative insect vector could become infected with the HLB-phytoplasma. In SPS, sunn hemp (Crotalaria juncea L.) is a major, widely distributed cover crop. A 16Sr group IX phytoplasma was detected in sunn hemp plants with witches’ broom and virescence symptoms, and was shown to have 16Sr DNA sequences and ribosomal protein gene sequences with 100% identity to the corresponding sequences of the sweet orange HLB-phytoplasma. Transmission electron microscopy revealed the presence of phytoplasma cells in the phloem sieve tubes of infected C. juncea stalks. These results were taken as evidence that the sunn hemp phytoplasma and the sweet orange HLB-phytoplasma were identical. Scaphytopius marginelineatus, a leafhopper frequently found in sweet orange orchards, was shown to acquire the HLB-phytoplasma efficiently from affected sunn hemp plants, but acquisition from, and transmission rates to, sweet orange were very low. On the whole, these data suggest that (i) sunn hemp is a major source of inoculum of the HLB-phytoplasma, (ii) S. marginelineatus becomes infected on sunn hemp and transmits the phytoplasma to sweet orange, and (iii) transmission from sweet orange to sweet orange occurs only rarely, if at all. 16Sr group IX phytoplasmas, very closely related to the SPS HLB-phytoplasma, have also been detected in citrus in Minas Gerais and Bahia states (Brazil) and Mexico.

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.

Citrus are an important subsistence and commercial crop across the South and Central Pacific. Unfortunately, the spread of plant material has contributed to the spread of citrus pathogens, such as Citrus tristeza virus (CTV). In this study, we examined the incidence and diversity of CTV strains present in both New Zealand, and island nations of the South and Central Pacific, and found that all presently described strains are present, and exist and complex mixtures of strains. Phylogenetic analysis suggests little difference in strain diversity between locations, suggesting extensive movement of infected planting material occurred in the past.

Huanglongbing (HLB) is considered the most devastating disease of citrus. The bacterium and vector associated with HLB in Florida are Candidatus Liberibacter asiaticus and Diaphorina citri (Asian citrus psyllid), respectively. D. citri is positively phototropic, and higher populations have been found along edges of orchards exposed to the sun. A survey was designed to determine if D. citri adult populations along edges of orchards varied according to time-of-day and time-of-year in relation to the azimuth of the sun. The survey was conducted twice. Citrus orchards, each divided into 9 sampling areas, were surveyed for D. citri via stem-tap sampling. Orchards were sampled 3 times per day (near sunrise, solar noon, and sunset) and 4 times per year (near the summer solstice, autumnal equinox, winter solstice, and vernal equinox). Time-of-year and sampling area significantly affected psyllid counts (P = 0.0518 and 0.0630, respectively). D. citri adults were most prevalent during the summer solstice sampling period. No overall significant time-of-day effect was observed (P > 0.6). Localization of adult D. citri in sampled citrus orchards did not significantly change in relation to time-of-year (P = 0.0907). Linear mixed regression was used to fit a quadratic equation to log D. citri abundance data in relation to elevation-corrected azimuth at the time of sampling; the fitted model was significant and predicted log D. citri abundance to exhibit a concave-up pattern with increasing elevation-corrected azimuth. This relationship represented in a new form how population counts of D. citri adults in Florida were greatest during the summer.

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.

Historically, decline (or tristeza) has been a devastating disease of citrus caused by Citrus tristeza virus (CTV). Decline is a man-made disease based largely on propagation of sweet orange, grapefruit, and mandarins on the sour orange rootstock. In Florida, the major problem caused by CTV has been decline, since severe stem-pitting isolates are absent. Although this disease can be controlled by using alternative rootstocks, there are soils in which all other rootstock choices are less desirable in terms of fruit quality and yield. A major goal has been to develop measures that allow growers to use the sour orange rootstock in the presence of CTV. Florida has 2 predominant strains of CTV, a decline (T36) strain and a non-decline strain (T30). A first step was to map the viral determinant that induces decline. This was done by creating hybrids with T30 sequences substituted into T36 to identify sequences correlated with loss of decline symptoms. This project was delayed considerably because greenhouse assays to definitively assay decline symptoms did not work. In order to examine decline in field trees, a permit from the US Department of Agriculture Biotechnology Regulatory Service was obtained to test the recombinant-DNA-produced virus hybrids. This permit required that we test small trees inoculated with CTV at the time of planting. Under these conditions, those constructs that retained the T36 p23 and 3ʹ non-translated sequences induced greater amounts of stunting.

The etiology of xyloporosis, a disease that has severe effects on citrus trees grafted onto certain citrus rootstocks, was enigmatic for a long time. Symptoms on test hosts following transmission through grafting suggested that it was synonymous with citrus cachexia, a disease that mainly affects mandarin trees. Recent molecular studies have confirmed that certain Hop stunt viroid (HSVd) isolates induce cachexia and xyloporosis symptoms in disease-sensitive citrus hosts. These HSVd infections are mostly symptomless in numerous Near East and Western Mediterranean fruit trees and grapevines; including plants widely cultivated in those regions for several millennia, long before the emergence of xyloporosis and cachexia as diseases of citrus trees. The present review tracks historical changes in citrus propagation practices and the pathological consequences of those changes that contributed to the emergence of xyloporosis as an economically significant disease of citrus trees grafted onto Palestinian sweet lime rootstocks. The take-home message of these accounts is the need for close cooperation between plant scientists, plant protection scientists, and growers to ensure that changes and proposed improvements in horticultural and plant protection practices are subjected to comprehensive risk-assessment analyses.

Phytophthora spp. are present in nearly all citrus groves in Florida and Brazil and phytophthora-induced diseases, especially foot and root rot, have the potential to cause economically important crop losses. Disease-related losses due to root rot are difficult to estimate because fibrous root damage and yield loss are not always directly proportional. Challenges from phytophthora diseases have been addressed in both countries by enacting phytosanitary requirements for production of pathogen-free nursery trees in enclosed structures, propagated from indexed and certified pathogen-free sources, in conjunction with several other cultural management practices. In Florida groves, a statewide soil sampling program provides growers with soil propagule counts to estimate the damage that Phytophthora spp. are causing to fibrous roots. The results can be used along with rootstock tolerance, soils, topography, irrigation, and drainage to make a decision for the need to treat with fungicides in addition to modification of cultural managements. Huanglongbing (HLB), caused by the psyllidtransmitted bacterium Candidatus Liberibacter asiaticus (Las), was detected in Brazil and Florida in the mid-2000s. Given the increasing incidence of HLB and deterioration of root density due to Las damage, research experiences and current phytophthora data trends suggest the need for more comprehensive management of root health by reducing the impact of abiotic and biotic stresses, including the interaction with Phytophthora spp.

Postbloom fruit drop (PFD), caused primarily by Colletotrichum acutatum, is a serious disease annually in the humid tropical citrus areas of the Americas and occurs more sporadically in the humid subtropics. The fungus infects flowers of all citrus species producing orange-brown lesions on petals that result in abscission of the fruitlets leaving the persistent calyx and floral disk attached to the twigs. C. acutatum also causes Key lime anthracnose and is morphologically identical to PFD, but the strains can be differentiated by molecular means and pathogenicity tests. C. acutatum produces abundant conidia on infected petals that are dispersed primarily by rain splash. After the bloom season, the fungus persists as appressoria on persistent calyces and other vegetative plant parts. Those appressoria are stimulated to germinate by flower extracts and produce secondary conidia to initiate a new disease cycle. Some cultural measures are useful in reducing disease severity, but control is based principally on application of fungicides. Benzimidazole fungicides and captafol were the best materials in the past but are no longer available. The QoI fungicides, folpet and tebuconazole + trifloxystrobin are the most effective among currently available materials. Forecasting systems based primarily on the availability of inoculum and recent rainfall were developed to aid growers on timing fungicide applications. The expert system, PFD-FAD, based on environmental conditions, inoculum, disease history, and varietal susceptibility is the most effective means currently available for scheduling fungicide applications.

Abstract First reported in 1896, psorosis was the first citrus disease proven to be graft transmissible and also the first for which eradication and budwood certification programs were launched to prevent its economic damage. For many years psorosis etiology remained elusive and only in 1986 it was associated with the presence of virus-like particles in infected plants. However, in the last two decades a virus with unusual morphology (Citrus psorosis virus, CPsV) was characterized and closely associated with psorosis disease as previously defined by field symptoms and by biological indexing in sensitive indicator plants. With a tripartite, negative-sense, RNA genome and a ~48 kDa coat protein, CPsV, the presumed causal agent of psorosis, is the type member of the genus Ophiovirus, within the new family Ophioviridae. Availability of the complete genomic sequence of two CPsV isolates and partial sequences of many others has enabled i) setting up rapid and sensitive RNA-based detection methods, ii) testing different citrus and relatives for resistance to CPsV, iii) identification of the two components (psorosis A and psorosis B) traditionally associated with non-scaled and scaled bark inoculum, respectively, from psorosis-infected plants and study their interactions, iv) analysis of genetic variation and evolutionary forces shaping the CPsV populations, v) preliminary studies on the interactions between virus and host factors and vi) development of transgenic citrus plants expressing variable degrees of resistance to CPsV. In summary, 120 years after the first report on psorosis we start seeing a pale light at the end of the tunnel.

The high health status of Australian citrus germplasm has been maintained largely due to a successful quarantine system and propagation scheme. Most endemic graft transmissible diseases are rarely observed in Australian orchards due to the use of high health status propagation material supplied by Auscitrus. Citrus tristeza virus is present throughout the citrus growing areas although mild strain cross protection has been effectively managing grapefruit stem pitting in white grapefruit varieties for over 40 years. However, diseases like huanglongbing and canker are ever present threats to the biosecurity of the Australian citrus industry. The introduction of mandatory nursery registration and compulsory use of pathogen tested propagation material would provide greater security to the industry in the face of increasing biosecurity threats.