Rodrigo P. P. Almeida

Living in two Worlds: The Plant and Insect Lifestyles of Xylella fastidiosa

Diseases caused by Xylella fastidiosa have attained great importance worldwide as the pathogen and its insect vectors have been disseminated. Since this is the first plant pathogenic bacterium for which a complete genome sequence was determined, much progress has been made in understanding the process by which it spreads within the xylem vessels of susceptible plants as well as the traits that contribute to its acquisition and transmission by sharpshooter vectors. Although this pathogen shares many similarities with Xanthomonas species, such as its use of a small fatty acid signal molecule to coordinate virulence gene expression, the traits that it utilizes to cause disease and the manner in which they are regulated differ substantially from those of related plant pathogens. Its complex lifestyle as both a plant and insect colonist involves traits that are in conflict with these stages, thus apparently necessitating the use of a gene regulatory scheme that allows cells expressing different traits to co-occur in the plant.

Use of a Green Fluorescent Strain for Analysis of Xylella fastidiosa Colonization of Vitis vinifera

ABSTRACT Xylella fastidiosa causes Pierces disease of grapevine as well as several other major agricultural diseases but is a benign endophyte in most host plants. X. fastidiosa colonizes the xylem vessels of host plants and is transmitted by xylem sap-feeding insect vectors. To understand better the pattern of host colonization and its relationship to disease, we engineered X. fastidiosa to express a green fluorescent protein (Gfp) constitutively and performed confocal laser-scanning microscopic analysis of colonization in a susceptible host, Vitis vinifera. In symptomatic leaves, the fraction of vessels colonized by X. fastidiosa was fivefold higher than in nearby asymptomatic leaves. The fraction of vessels completely blocked by X. fastidiosa colonies increased 40-fold in symptomatic leaves and was the feature of colonization most dramatically linked to symptoms. Therefore, the extent of vessel blockage by bacterial colonization is highly likely to be a crucial variable in symptom expression. Intriguingly, a high proportion (>80%) of colonized vessels were not blocked in infected leaves and instead had small colonies or solitary cells, suggesting that vessel blockage is not a colonization strategy employed by the pathogen but, rather, a by-product of endophytic colonization. We present evidence for X. fastidiosa movement through bordered pits to neighboring vessels and propose that vessel-to-vessel movement is a key colonization strategy whose failure results in vessel plugging and disease.

Vector Transmission of Xylella fastidiosa: Applying Fundamental Knowledge to Generate Disease Management Strategies

Abstract Xylella fastidiosa is a xylem-limited bacterium transmitted to plants by xylem sap-feeding insects. This pathogen has a wide host range, causing disease in crops such as grape, citrus, almond, and coffee; ornamental plants; and trees. Sharpshooter leafhoppers are the major vectors of X. fastidiosa to crops of economic importance. Transmission characteristics include the lack of a latent period, no transstadial or transovarial transmission, persistence in adults, and multiplication in the foregut of vectors. Various factors influence vector transmission of X. fastidiosa, including the distribution and density of bacterial populations in host plants, insect host range and plant preference, season of inoculation, and climatic conditions. The ecology of vectors can affect epidemics, as demonstrated by the large increase in Pierce’s disease of grapevine incidence in California after the introduction of Homalodisca coagulata (Say). Disease control strategies should incorporate basic knowledge of transmission biology, vector ecology, and other interactions involved in X. fastidiosa diseases. We discuss basic aspects of X. fastidiosa transmission by vectors, the ecology of insects in relation to transmission and disease spread, and how basic research can be applied to the development of management strategies for a X. fastidiosa disease.

Biological traits of Xylella fastidiosa strains from grapes and almonds.

ABSTRACT Xylella fastidiosa is a xylem-limited bacterium that causes various diseases, among them Pierces disease of grapevine (PD) and almond leaf scorch (ALS). PD and ALS have long been considered to be caused by the same strain of this pathogen, but recent genetic studies have revealed differences among X. fastidiosa isolated from these host plants. We tested the hypothesis that ALS is caused by PD and ALS strains in the field and found that both groups of X. fastidiosa caused ALS and overwintered within almonds after mechanical inoculation. Under greenhouse conditions, all isolates caused ALS and all isolates from grapes caused PD. However, isolates belonging to almond genetic groupings did not cause PD in inoculated grapes but systemically infected grapes with lower frequency and populations than those belonging to grape strains. Isolates able to cause both PD and ALS developed 10-fold-higher concentrations of X. fastidiosa in grapes than in almonds. In the laboratory, isolates from grapes overwintered with higher efficiency in grapes than in almonds and isolates from almonds overwintered better in almonds than in grapes. We assigned strains from almonds into groups I and II on the basis of their genetic characteristics, growth on PD3 solid medium, and bacterial populations within inoculated grapevines. Our results show that genetically distinct strains from grapes and almonds differ in population behavior and pathogenicity in grapes and in the ability to grow on two different media.

Mealybug transmission of Grapevine leafroll viruses: an analysis of virus-vector specificity.

To understand ecological factors mediating the spread of insect-borne plant pathogens, vector species for these pathogens need to be identified. Grapevine leafroll disease is caused by a complex of phylogenetically related closteroviruses, some of which are transmitted by insect vectors; however, the specificities of these complex virus-vector interactions are poorly understood thus far. Through biological assays and phylogenetic analyses, we studied the role of vector-pathogen specificity in the transmission of several grapevine leafroll-associated viruses (GLRaVs) by their mealybug vectors. Using plants with multiple virus infections, several virus species were screened for vector transmission by the mealybug species Planococcus ficus and Pseudococcus longispinus. We report that two GLRaVs (-4 and -9), for which no vector transmission evidence was available, are mealybug-borne. The analyses performed indicated no evidence of mealybug-GLRaV specificity; for example, different vector species transmitted GLRaV-3 and one vector species, Planococcus ficus, transmitted five GLRaVs. Based on available data, there is no compelling evidence of vector-virus specificity in the mealybug transmission of GLRaVs. However, more studies aimed at increasing the number of mealybug species tested as vectors of different GLRaVs are necessary. This is especially important given the increasing importance of grapevine leafroll disease spread by mealybugs in vineyards worldwide.

Transmission of Grapevine leafroll-associated virus 3 by the Vine Mealybug (Planococcus ficus)

Grapevine leafroll disease is caused by grapevine leafroll-associated viruses (GLRaVs). Within this virus complex, GLRaV-3 is the predominant species in the world. Several GLRaVs have been shown to be transmitted from vine to vine by mealybugs although a detailed characterization of transmission biology is lacking. The introduction of the vine mealybug (Planococcus ficus) in California and other regions of the world may result in increasing disease incidence of established GLRaVs. We studied the characteristics of GLRaV-3 transmission by the vine mealybug. Our results indicate that the vine mealybug transmits GLRaV-3 in a semipersistent manner. First instars were more efficient vectors than adult mealybugs. GLRaV-3 transmission lacked a latent period in the vector. Virus transmission occurred with a 1-h acquisition access period (AAP) and peaked with a 24-h AAP. Mealybugs inoculated GLRaV-3 with a 1-h inoculation access period (IAP), and transmission efficiency increased with longer plant access period up to 24 h, after which transmission rate remained constant. After an AAP of 24 h, mealybugs lost GLRaV-3 and infectivity 4 days after virus acquisition. In addition, GLRaV-3 was not transovarially transmitted from infected females to their progeny as detected by reverse transcription polymerase chain reaction. In summary, we systematically analyzed transmission parameters of GLRaV-3 by the vine mealybug and showed that transmission of this virus occurs in a semipersistent manner. This research fills in important gaps in knowledge of leafroll virus transmission, which is critical for development of leafroll disease management practices.

Grapevine leafroll-associated virus 3

Grapevine leafroll disease (GLD) is one of the most important grapevine viral diseases affecting grapevines worldwide. The impact on vine health, crop yield, and quality is difficult to assess due to a high number of variables, but significant economic losses are consistently reported over the lifespan of a vineyard if intervention strategies are not implemented. Several viruses from the family Closteroviridae are associated with GLD. However, Grapevine leafroll-associated virus 3 (GLRaV-3), the type species for the genus Ampelovirus, is regarded as the most important causative agent. Here we provide a general overview on various aspects of GLRaV-3, with an emphasis on the latest advances in the characterization of the genome. The full genome of several isolates have recently been sequenced and annotated, revealing the existence of several genetic variants. The classification of these variants, based on their genome sequence, will be discussed and a guideline is presented to facilitate future comparative studies. The characterization of sgRNAs produced during the infection cycle of GLRaV-3 has given some insight into the replication strategy and the putative functionality of the ORFs. The latest nucleotide sequence based molecular diagnostic techniques were shown to be more sensitive than conventional serological assays and although ELISA is not as sensitive it remains valuable for high-throughput screening and complementary to molecular diagnostics. The application of next-generation sequencing is proving to be a valuable tool to study the complexity of viral infection as well as plant pathogen interaction. Next-generation sequencing data can provide information regarding disease complexes, variants of viral species, and abundance of particular viruses. This information can be used to develop more accurate diagnostic assays. Reliable virus screening in support of robust grapevine certification programs remains the cornerstone of GLD management.

Vertical Transmission of a Pentatomid Caeca-Associated Symbiont

Abstract We present molecular data for an endosymbiont of the insect family Pentatomidae, located in the gastric caeca of Nezara viridula (L.) (Hemiptera: Pentatomidae) stink bugs. Restriction fragment length polymorphism and polymerase chain reaction analysis suggest that this bacterium is consistently present in caeca of N. viridula from a variety of geographic locations. The bacterium is present in different midgut sections in nymphs versus adults. The bacterium also was detected on eggshells after nymphs had hatched but not in ovarioles, suggesting oral rather than transovarial transmission. Surface sterilization of egg masses generated aposymbiotic insects. Aposymbiotic individuals reached the adult stage, females laid viable eggs, and the offspring remained aposymbiotic in the following generation. No clear fitness decrease was observed in aposymbiotic individuals over two generations. Phylogenetic analysis of a partial 16S rRNA data set with 21 Gammaproteobacteria suggested the inadequacy of neighbor-joining and maximum parsimony models to account for homoplasy apparent in a molecular data set, including a range of insect endosymbionts. Maximum likelihood-based analysis suggests that the N. viridula endosymbiont is closely related to a caeca-associated symbiont found in another stink bug family (Plataspidae). The high AT content of the symbiont’s 16S rRNA in relation to other insect endosymbionts, its location in the midgut of the host insect, oral transmission, and survival of aposymbiotic individuals suggest this symbiosis may be recently established.

Multiplication and movement of a citrus strain of Xylella fastidiosa within sweet orange

Populations of cultivable cells of a citrus variegated chlorosis (CVC) disease strain of Xylella fastidiosa in stems and leaf veins of sweet orange (Citrus sinensis (L.) Osbeck) seedlings were estimated by dilution plating at 1, 2, 4, 8, and 16 weeks after needle inoculation. Cell populations ranged from log 4 to log 5 CFU/g of tissue after 1 week and increased to log 5 to log 7 CFU/g (median log 6) after 8 to 16 weeks. Recovery of greater than log 5 CFU/g from stem nodes distal to the inoculation site indicated systemic movement of the bacteria. Foliar symptoms in inoculated seedlings first appeared after 8 weeks. Population estimates from leaf veins of CVC-affected trees in citrus groves were in the same range but slightly lower (average log 5.8 CFU/g). X. fastidiosa was isolated from citrus more efficiently in periwinkle wilt-GelRite (PWG) and periwinkle wilt (PW) media than in charcoal-yeast extract with ACES buffer (BCYE) medium The relatively lower populations of cultivable cells of X. fastidiosa in citrus with CVC symptoms, compared with those reported in grapevines with Pierces disease, suggest that most cells of X. fastidiosa within symptomatic citrus may be dead, explaining in part the low rates of vector transmission from citrus to citrus.

Biology and Management of Mealybugs in Vineyards

Economic losses resulting from vineyard mealybug infestations have increased dramatically during the past decade. In response, there has been a cosmopolitan effort to improve control strategies and better understand mealybug biology and ecology, as well as their role as vectors of plant pathogens. Mealybugs are named for the powdery secretions covering their bodies. The most important vineyard mealybugs belong to the subfamily Pseudococcinae (Hardy et al. 2008). Although numerous mealybug species are found in vineyards, this chapter will cover only those that have risen to the level of primary pest. These are the grape mealybug, Pseudococcus maritimus (Ehrhorn), obscure mealybug, Pseudococcus viburni (Signoret), long-tailed mealybug, Pseudococcus longispinus (Targioni Tozzetti), citrophilus mealybug, Pseudococcus calceolariae (Maskell), vine mealybug, Planococcus ficus (Signoret), citrus mealybug, Planococcus citri (Risso), pink hibiscus mealybug, Maconellicoccus hirsutus (Green), and the newly identified Gills mealybug, Ferrisia gilli Gullan. Meanwhile in Brazil and India, Dysmicoccus brevipes (Cockerell) and Xenococcus annandalei Silvestri respectively, feed on vine roots. Collectively, these species will be referred to as the vineyard mealybugs, although their host range is diverse and many are pests of other agricultural crops and ornamental plants (McKenzie 1967; Ben-Dov 1995).