Chandrika Ramadugu

Trailers Transporting Oranges to Processing Plants Move Asian Citrus Psyllids

ABSTRACT Huanglongbing (citrus greening) is one of the most serious diseases of citrus. Movement of the disease occurs as a result of natural transmission by the insect vector and by movement of infected plant material. We demonstrate here that Diaphorina citri Kuwayama, the vector of citrus greening pathogens, can be transported in trailers of unprocessed fruit. Several samples of D. citri collected from trailers of oranges were positive for citrus greening pathogens, indicating that the disease can spread widely with the movement of the mature fruit as a result of moving the vectors. While movement of disease and the vector through plant materials into new areas has been recognized, our findings emphasize the need to evaluate the importance of long distance movement of psyllids on unprocessed fruit, even in the absence of vegetative tissue.

A six nuclear gene phylogeny of Citrus (Rutaceae) taking into account hybridization and lineage sorting.

Background Genus Citrus (Rutaceae) comprises many important cultivated species that generally hybridize easily. Phylogenetic study of a group showing extensive hybridization is challenging. Since the genus Citrus has diverged recently (4–12 Ma), incomplete lineage sorting of ancestral polymorphisms is also likely to cause discrepancies among genes in phylogenetic inferences. Incongruence of gene trees is observed and it is essential to unravel the processes that cause inconsistencies in order to understand the phylogenetic relationships among the species. Methodology and Principal Findings (1) We generated phylogenetic trees using haplotype sequences of six low copy nuclear genes. (2) Published simple sequence repeat data were re-analyzed to study population structure and the results were compared with the phylogenetic trees constructed using sequence data and coalescence simulations. (3) To distinguish between hybridization and incomplete lineage sorting, we developed and utilized a coalescence simulation approach. In other studies, species trees have been inferred despite the possibility of hybridization having occurred and used to generate null distributions of the effect of lineage sorting alone (by coalescent simulation). Since this is problematic, we instead generate these distributions directly from observed gene trees. Of the six trees generated, we used the most resolved three to detect hybrids. We found that 11 of 33 samples appear to be affected by historical hybridization. Analysis of the remaining three genes supported the conclusions from the hybrid detection test. Conclusions We have identified or confirmed probable hybrid origins for several Citrus cultivars using three different approaches–gene phylogenies, population structure analysis and coalescence simulation. Hybridization and incomplete lineage sorting were identified primarily based on differences among gene phylogenies with reference to null expectations via coalescence simulations. We conclude that identifying hybridization as a frequent cause of incongruence among gene trees is critical to correctly infer the phylogeny among species of Citrus.

Incidence of Huanglongbing-Associated ‘Candidatus Liberibacter Asiaticus’ in Diaphorina citri (Hemiptera: Psyllidae) Collected from Plants for Sale in Florida

ABSTRACT The Asian citrus psyllid, Diaphorina citri Kuwayama, was reported for the first time in Florida in June 1998, and huanglongbing (HLB, citrus greening), vectored by D. citri, was detected in Florida for the first time in Aug 2005. In Florida, the only known HLB pathogen is ‘Candidatus Liberibacter asiaticus’ (Las). After HLB was known to be established in Florida, the psyllid vectors found in regulatory samples from plants for sale were tested for the pathogen by real-time qPCR. Approximately 1,200 regulatory samples were tested between Aug 2005 and Aug 2009. Samples came from venues in 44 of Floridas 67 counties. Most of the samples came from citrus, but about 11% came from Murraya exotica, a popular ornamental plant and close relative of citrus. Approximately 9.7% of the psyllid samples tested were positive for Las. Numbers of samples and proportion of positive samples varied by year and by county.

Long-Term Field Evaluation Reveals Huanglongbing Resistance in Citrus Relatives

Citrus huanglongbing (HLB) is a destructive disease with no known cure. To identify sources of HLB resistance in the subfamily Aurantioideae to which citrus belongs, we conducted a six-year field trial under natural disease challenge conditions in an HLB endemic region. The study included 65 Citrus accessions and 33 accessions belonging to 20 other closely related genera. For each accession, eight seedling trees were evaluated. Based on quantitative polymerase chain reaction analysis of the pathogen titers and disease symptoms, eight disease-response categories were identified. We report two immune, six resistant, and 14 tolerant accessions. Resistance and tolerance observed in different accessions may be attributed to a multitude of factors, including psyllid colonization ability, absence of pathogen multiplication, transient replication of the bacterium, lack of pathogen establishment in the plant, delayed infection, or recovery from infection. Most citrus cultivars were considered susceptible: 15 citrons, lemons, and limes retained leaves in spite of the disease status. Resistance and high levels of field tolerance were observed in many noncitrus genera. Disease resistance/tolerance was observed in Australian citrus relative genera Eremocitrus and Microcitrus, which are sexually compatible with citrus and may be useful in future breeding trials to impart HLB resistance to cultivated citrus.

Changes in Anthocyanin Production during Domestication of Citrus

Ruby, a regulatory gene encoding a MYB transcription factor, is essential for anthocyanin production, and differences in its activity determine most of the natural variation in pigmentation in Citrus and related genera. Mandarin (Citrus reticulata), citron (Citrus medica), and pummelo (Citrus maxima) are important species of the genus Citrus and parents of the interspecific hybrids that constitute the most familiar commercial varieties of Citrus: sweet orange, sour orange, clementine, lemon, lime, and grapefruit. Citron produces anthocyanins in its young leaves and flowers, as do species in genera closely related to Citrus, but mandarins do not, and pummelo varieties that produce anthocyanins have not been reported. We investigated the activity of the Ruby gene, which encodes a MYB transcription factor controlling anthocyanin biosynthesis, in different accessions of a range of Citrus species and in domesticated cultivars. A white mutant of lemon lacks functional alleles of Ruby, demonstrating that Ruby plays an essential role in anthocyanin production in Citrus. Almost all the natural variation in pigmentation by anthocyanins in Citrus species can be explained by differences in activity of the Ruby gene, caused by point mutations and deletions and insertions of transposable elements. Comparison of the allelic constitution of Ruby in different species and cultivars also helps to clarify many of the taxonomic relationships in different species of Citrus, confirms the derivation of commercial varieties during domestication, elucidates the relationships within the subgenus Papeda, and allows a new genetic classification of mandarins.

The Origin of Oranges: A Multi-Locus Phylogeny of Rutaceae Subfamily Aurantioideae

Abstract The phylogeny of Rutaceae subfamily Aurantioideae has previously been estimated only using plastid and repetitive nuclear sequences. We added sequences of two low copy nuclear loci to allow further diagnosis of phenomena that may mislead phylogenetic inference. After testing for patterns expected under recombination, positive selection, and hybridization, we excluded data sets or sequences accordingly and then inferred the species tree using the multispecies coalescent. We then reconstructed the ancestral area using parsimony and the dispersal-extinction-cladogenesis model to test the hypothesis that Citrus s. l. may have originated in Australasia and migrated or rafted to Eastern Asia. The ancestral area of Citrus s. l. inferred under either method and several models was west of Wallaces line. Therefore, Citrus s. l. did not appear to have rafted west on what became the Halmahera Islands (Indonesia). Our findings are also consistent with previously reported ages for the origin of this group that may be too young to have allowed this rafting. The species tree is well resolved and largely consistent with previous molecular phylogenies, especially those using chloroplast sequences.

Apparent Tolerance to Huanglongbing in Citrus and Citrus-related Germplasm

Abstract. In a Fort Pierce, FL, field planting, plant growth, and Huanglongbing (HLB) severity were assessed as indicators of HLB tolerance on progenies of 83 seed-source accessions of Citrus and Citrus relatives mainly from the Riverside, CA, genebank. The HLB-associated pathogen [Candidatus Liberibacter asiaticus (CLas)] and vector [asian citrus psyllid (ACP), Diaphorina citri] were abundant, and trees were naturally challenged for 6 years before metrics (leaf mottle, percent canopy mottle, overall health, canopy density, canopy width, canopy height, and trunk diameter) were collected in Oct. and Nov. 2015. The healthiest trees with low or no HLB symptoms were distant citrus relatives: Balsamocitrus dawei, Bergera koenigii, Casimiroa edulis, Clausena excavata, Murraya paniculata, and one accession of Severinia buxifolia. Within Citrus, most of the healthiest trees with densest canopies, little leaf loss, and greater growth were those with pedigrees that included Citrus medica (citron). These included progenies of Citrus hybrid (‘Limon Real’), Citrus limetta, Citrus limettioides, Citrus limonia, C. medica, Citrus volkameriana, and some Citrus limon accessions. Trees in this category exhibited distinct leaf-mottle characteristic of HLB and substantial pathogen titers, but maintained dense canopies and exhibited good growth. Trees from seed-source accessions in the genus Citrus without citron in their background were generally among the least healthy overall with less dense canopies. The exceptions were progenies of two Citrus aurantium accessions, which were markedly healthier than progenies of other Citrus seed-source accessions not derived from citron. Linear regression analysis, between metrics collected and pedigree of seed parent, indicated that percentage of citron in the pedigree significantly correlated with measures of tolerance. Although no commercial Citrus genotypes yielded progenies with strong HLB resistance, in this field experiment several progenies maintained dense canopies and good growth, and may be useful for breeding HLB tolerant cultivars.