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Australasian Plant Pathology, 2006, 35, 129–146

Phytoplasma diseases in sub-tropical and tropical Australia C. StretenA,B and K. S. GibbA A Charles

Darwin University, Darwin 0909, Australia. author. Email: [email protected]

B Corresponding

Abstract. Phytoplasmas are phloem-limited plant pathogens that have been identified in over 1000 plant species worldwide. Outbreaks of the phytoplasma-related disease, papaya dieback, has resulted in 10–100% crop losses in south-east Queensland and Western Australia. Strawberry lethal yellows and green petal disease outbreaks in Queensland have led to 10–50% of strawberry runners being destroyed. Lucerne yellows disease has been reported to cause an annual loss of AU$7 million to the lucerne seed industry. Disease surveys in Australia have increased our understanding of phytoplasma diseases in Australia and these fastidious organisms have been detected in ∼70 native and introduced plant species. The majority of the Australian phytoplasmas are assigned to the 16SrII group, however, a member of the 16SrXII group is more commonly associated with economically important diseases in Australia such as strawberry lethal yellows, papaya dieback and grapevine yellows. These phytoplasma diseases have been diagnosed using PCR primers specific for their 16S rRNA gene. Screening hundreds of samples using PCR is time consuming and expensive so current and future studies are characterising an Australian phytoplasma genome and identifying suitable targets for the development of a more rapid diagnostic test for phytoplasmas.

Introduction History of phytoplasma research—worldwide Symptoms of stunting, yellowing, phyllody and virescence were reported in the early 1900s (Lee et al. 2000). An organism of viral origin was thought to be associated with these plant diseases because the organism could be transmitted to healthy plants by grafting or insect vectors but could not be cultured in vitro and it passed through bacteria-proof filters (Lee et al. 2000). These diseases were collectively referred to as the ‘yellows group’ (Bowyer et al. 1969). In the mid 1960s, Doi et al. (1967) observed pleomorphic bodies in the phloem of diseased mulberry plants. Microscopy studies showed that the pleomorphic bodies range in size from 200–1000 nm (McCoy et al. 1989) and are surrounded by a triple layered single unit membrane instead of a cell wall (Lee and Davis 1992). The internal ultrastructure of bodies consists of strands that are thought to be DNA and granules that are the size of prokaryotic ribosomes (Lee and Davis 1992). The bodies also disappeared from the phloem of diseased mulberry plants after tetracycline treatment (Ishiie et al. 1967). These characteristics resemble those of the mycoplasmas which led to these organisms being assigned to the class Mollicutes (Doi et al. 1967) and they were assigned the name ‘mycoplasma-like organisms’ or MLOs (Lee and Davis 1992). The term MLO was used until the trivial name ‘phytoplasma’ was proposed by the Phytoplasma © Australasian Plant Pathology Society 2006

Working Team in 1994 (International Committee Systematic Bacteriology Subcommittee Taxonomy Mollicutes 1993, 1997). Phytoplasmas also have genomic characteristics that are common to members of the class Mollicutes (Seemüller et al. 1998) (Table 1). Phytoplasmas have a small genome with a low G+C content (23–35%) (Kollar and Seemüller 1989) and genome sizes range from 530 to 1185 kb (Neimark and Kirkpatrick 1993; Marcone et al. 1999) (Table 1). These findings supported the assignment of phytoplasmas to the class Mollicutes. Initial work on the 16S rRNA gene of the aster yellows phytoplasma showed that this phytoplasma represented a distinct monophyletic clade within the class Mollicutes (Lim and Sears 1992; Gundersen et al. 1994; Schneider et al. 1995). Furthermore, sequence analysis revealed that the phytoplasmas are more closely related to the Acholeplasma species, A. palmae and A. laidlawii, than to Mycoplasma species (Sears and Kirkpatrick 1994). The relationship between the phytoplasmas and Acholeplasma spp. was supported by comparative analysis of the ribosomal protein (rp) encoding genes (rps19-rpl22-rps3) (Kuske and Kirkpatrick 1992; Lim and Sears 1991, 1992). Diagnosis and classification Phytoplasmas were originally detected by electron microscopy of ultra-thin sections from symptomatic plants (Doi et al. 1967; Greber and Gowanlock 1979). In the 10.1071/AP06004

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Australasian Plant Pathology

C. Streten and K. S. Gibb

Table 1. Characteristics of phytoplasmas, spiroplasmas and phloem-limited bacteria Character Cell wall Genome size G+C content Localisation Antibiotic sensitivity Transmission Culture in vitro A B

Phloem-limited bacteria

Organism Phytoplasmas

Spiroplasmas

PresentA 3–5 kbA — Phloem, intracellularA Tetracycline, PenicillinA Circulative, leafhoppers, psyllids, aphidsA No

Absent 0.53–1.2 kb 23–35% Phloem, intracellular Tetracycline Circulative, leafhoppers, planthoppers, psyllids No

AbsentB 1.8 kb 25–31%A Phloem, intracellularA TetracyclineB Circulative, leafhoppers, psyllids Yes

Cousin (1999). Behncken (1983).

1980s, enzyme-linked immunosorbent assays (ELISA) using monoclonal and polyclonal antibodies were developed for the detection of phytoplasmas (Clark et al. 1983; Lin and Chen 1985; Lee and Davis 1993). While ELISA was suitable for the detection of phytoplasmas, it did not allow differentiation of types (Seemüller et al. 1998). In the late 1980s, DNA hybridisations and RFLP analysis of phytoplasma genomic DNA were used to detect and differentiate phytoplasmas (Kirkpatrick et al. 1987; Lee and Davis 1988; Deng and Hiruki 1991b; Lee et al. 1991). In the early 1990s, polymerase chain reaction (PCR) assays were developed to detect phytoplasmas (Deng and Hiruki 1991a; Ahrens and Seemüller 1992; Lee et al. 1993). RFLP analysis of 16S rRNA genes was used to differentiate phytoplasmas (Lee et al. 1998). Phytoplasma diagnostics are now routinely based on PCR amplification of 16S rRNA genes and RFLP analysis (Seemüller et al. 1998; Lee et al. 2000). Using these techniques, phytoplasmas are associated with diseases of over 1000 different plant species (McCoy et al. 1989; Seemüller et al. 1998). Phytoplasma diagnostics can be confounded by uneven distribution and low titre in the host plant (Constable et al. 2003). To increase the sensitivity of PCR assays, researchers have used nested PCR (Andersen et al. 1998a; Lee et al. 1994) and this approach was useful for detecting mixed phytoplasma ‘infections’ (Lee et al. 1994, 2000). Subsequently, researchers have tended to replace RFLP analysis with sequence analysis. All of these methods require well equipped laboratories, trained personnel and significant financial input, so there is an argument for developing specific antibody-based tests (Loi et al. 2002) which can be used in laboratories with only basic facilities. This approach is particularly useful for laboratories that are not equipped for molecular diagnostics. The inability to culture phytoplasma in vitro means that these organisms are not classified using the traditional criteria for culturable bacteria which are based on phenotypic and genotypic characteristics (Seemüller et al. 1998; Lee et al. 2000). As a result, phytoplasmas were originally classified on the basis of symptoms, host range and vector

specificity (Lee and Davis 1992; Seemüller et al. 1998; Lee et al. 2000). The problem with this system is that the same phytoplasma can cause different symptoms in different hosts, such as those observed for periwinkle, and different symptoms in the same host (strawberry) (Lee and Davis 1992; Padovan et al. 2000b). The introduction of DNA-based methods, southern hybridisations and RFLP analysis of phytoplasma genomic DNA revealed phytoplasma groups and subgroups (Deng and Hiruki 1991b; Lee et al. 1991). However, a DNAbased classification system was not available until researchers began to routinely study the 16S rRNA gene of phytoplasmas (Seemüller et al. 1998; Lee et al. 2000). Woese (1987) proposed that the 16S rRNA gene is a suitable phylogenetic marker for classifying prokaryotes because it is a universal gene for prokaryotes and contains variable and non variable regions which makes it suitable for taxonomic studies. Phytoplasmas have two 16S rRNA genes and both copies of this gene have been characterised for the onion yellows and Phormium yellow leaf phytoplasmas (Schneider and Seemüller 1994; Liefting et al. 1996; Jung et al. 2003). The two copies of the 16S rRNA genes isolated from the same phytoplasma vary by 0.27–0.1% (Liefting et al. 1996; Jung et al. 2003). RFLP analysis of 16S rRNA genes was used as a basis for a classification scheme for phytoplasmas (Lee et al. 1993, 1998; Schneider et al. 1993; Gundersen et al. 1994; Seemüller et al. 1994). This technique is used routinely to identify phytoplasmas, but RFLP analysis has largely been replaced by 16S rRNA gene nucleotide sequence comparisons for phylogeny studies (Seemüller et al. 1998; Lee et al. 2000). Members of the phytoplasma clade were differentiated into 12 subclades (16S rDNA group) through sequence analysis, and these groups correlated with those based on RFLP analysis of the 16S rRNA gene (Kuske and Kirkpatrick 1992; Seemüller et al. 1994; Lee et al. 1998). Kirkpatrick and Smart (1995) reported that phylogeny derived from the phytoplasma 16S–23S spacer region that is adjacent to the 16S rRNA gene is consistent with

Phytoplasma diseases in sub-tropical and tropical Australia

classification based on the 16S rRNA gene. Although groups differentiated by the 16S–23S region were consistent with those of the 16S rRNA gene, the authors proposed that the 16S–23S region is possibly more variable than the 16S rRNA gene so it may facilitate finer resolution of phylogenetic groupings (Kirkpatrick and Smart 1995). As a result, phytoplasmas are generally identified and assigned to 16SrI groups based on sequence analysis or RFLP analysis of the combined 16S rRNA gene and 16S–23S spacer region (Kirkpatrick and Smart 1995; Seemüller et al. 1998; Lee et al. 2000; IRPCM Phytoplasma/Spiroplasma Working Team— Phytoplasma taxonomy group 2004). Phytoplasmas are now differentiated into 15 phytoplasma 16Sr groups using RFLP or sequence analysis of the 16S rRNA gene and spacer region (Lee et al. 2000). There are five phytoplasmas which are not assigned to these phytoplasma groups (Seemüller et al. 1998; Lee et al. 2000). Phytoplasmas assigned to the same 16S rDNA group are generally designated the same species name because their 16S rRNA genes share more than 97.5% similarity (IRPCM Phytoplasma/Spiroplasma Working Team—Phytoplasma taxonomy group 2004). It has been recommended that a Candidatus Phytoplasma species name should be assigned to a single, unique phytoplasma 16S rRNA gene sequence (IRPCM Phytoplasma/Spiroplasma Working Team—Phytoplasma taxonomy group 2004). In addition, the committee recommended that phytoplasma strains which share greater that 97.5% 16S rRNA gene sequence similarity should be assigned the same species name except if there are host range, insect vector and serological data which support the separation of the phytoplasma into different species (IRPCM Phytoplasma/Spiroplasma Working Team— Phytoplasma taxonomy group 2004). Until recently, only three phytoplasma species had been assigned species names but all phytoplasma 16Sr groups are currently being assigned Candidatus Phytoplasma species names using the minimal taxonomic standards for uncultivated bacteria (IRPCM Phytoplasma/Spiroplasma Working Team—Phytoplasma taxonomy group 2004). Minimal taxonomic standards are used for the classification of phytoplasmas because these fastidious organisms have not been cultured, so Koch’s postulates cannot be proven. As a result, it is not possible to demonstrate phytoplasmas ‘cause’ a disease and so they are said to be ‘associated’ with a disease. However, a phytoplasma should only be considered to be associated with a disease if a significant relationship between organism and disease can be demonstrated. Differentiation studies of closely related phytoplasmas based on the 16S rRNA gene show no clear relationship between phytoplasma, host plant species or symptoms (Seemüller et al. 1998). Slight genomic differences that may account for the variation in symptoms and host specificity may not be detected because of the highly conserved nature of the 16S rRNA gene (Cilia et al. 1996; Ludwig and Schleifer


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1999). Furthermore, the 16S rRNA gene may not be useful for identifying subgroups within each phytoplasma 16SrI group (Gundersen et al. 1996; Marcone et al. 2000). Members of the same phytoplasma 16SrI group can also be differentiated using more variable regions or genes such as the tuf gene, ribosomal protein operon (rps19-rpl22-rps3), or nitroreductase (Jarausch et al. 1994, 2000; Gundersen et al. 1996; Liefting et al. 1998; Marcone et al. 2000; Padovan et al. 2000b). Phytoplasmas assigned to the 16SrI (aster yellows) and 16SrIII (X-disease and related phytoplasmas) can be divided into subgroups based on ribosomal protein (rp) and 16S rRNA gene sequences (Gundersen et al. 1996). Nine 16SrI and eight 16SrIII subgroups are identified by RFLP analysis of the ribosomal protein gene cluster (Gundersen et al. 1996; Jomantiene et al. 1998; Lee et al. 1998). These groupings are designated rr-rp subgroups (Gundersen et al. 1996; Jomantiene et al. 1998; Lee et al. 1998) and Gundersen et al. (1996) proposed that the rr-rp subgroups should be equivalent to subspecies. Marcone et al. (2000) reported that the tuf genes of phytoplasmas assigned to the 16SrI group are more conserved than the 16S rRNA genes. This was in contrast to members of the 16SrXII (stolbur) and 16SrX (apple proliferation) groups where the tuf genes are more variable than their 16S rRNA genes (Schneider et al. 1997; Berg and Seemüller 1999; Padovan et al. 2000b; Streten and Gibb 2005). Genetic variability is also observed between the nitroreductase genes of phytoplasmas assigned to the 16SrX group (Jarausch et al. 1994, 2000). It has been postulated that phytoplasma membraneassociated proteins are more variable than housekeeping proteins because the hydrophilic regions of these proteins are externally exposed and, therefore, under greater evolutionary pressure (Davis and Sinclair 1998; Blomquist et al. 2001; Barbara et al. 2002). A study of the immunodominant membrane protein genes of the European stone fruit yellows, peach yellow leaf roll and European pear decline phytoplasmas showed that the genetic variability between these phytoplasmas reflected the relationships based on 16S rRNA gene sequences (Morton et al. 2003). Morton et al. (2003) suggested that the greater genetic variability between these genes is possibly related to the host because the surface exposed proteins interact with plant and insect cells. Disease cycle Phytoplasmas are transmitted by phloem feeding leafhoppers (Cicadellidae), planthoppers (Fulgoridae) and psyllids (Psyllidae) through circulative and persistent transmission (Kirkpatrick 1989; Fletcher et al. 1998; Lee et al. 2000). An insect acquires the phytoplasma by feeding on the phloem of an infected plant (Lefol et al. 1993) (Fig. 1). The source plant can affect leafhopper acquisition of the phytoplasmas from the host (Bosco et al. 1997). The inability of vectors to acquire from a particular plant

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Latency period (2–6 weeks) Phytoplasma crosses insect gut wall, multiples in the haemolymph and salivary glands.

Inoculation (ⱖ1 hour) Insect transmits phytoplasma to another plant.

Acquisition (ⱖ1 hour) Insect acquires phytoplasma from phloem.

Plant latency (length depends on host and phytoplasma strain) Phytoplasma replicates in plant and symptoms develop.

Fig. 1. Disease cycle of phytoplasmas.

species may be due to plant metabolites which disrupt insect feeding or the insect’s feeding behaviour may vary depending upon the host plant (Bosco et al. 1997). For example, the leafhopper Empoasca vitis can feed from either the phloem or cortical parenchyma cells (Bosco et al. 1997). Alternatively, acquisition may be affected by the titre of the phytoplasma in the host plant (Bosco et al. 1997). After acquisition, the ingested phytoplasmas cross the gut wall of the insect and multiply in the haemolymph. From here, they move into the salivary glands where they continue to multiply (Lefol et al. 1993; Kirkpatrick 1989) (Fig. 1). The mechanisms involved in the movement of phytoplasmas across the insect membranes are poorly understood (Fletcher et al. 1998). Surface adhesions are proposed as having a role in phytoplasma transmission (Fletcher et al. 1998) and Lefol et al. (1995) provided indirect evidence that suggests the flavescence dorée phytoplasma attaches to specific receptors on the midgut and salivary glands of its vector, Scaphoideus littoralis. The involvement of receptors suggests that the phytoplasma itself contains the genetic information required for penetration of their insect vector (Garnier et al. 2001). To date, no phytoplasma proteins have been shown to be involved in colonisation of the insect. However, studies of Spiroplasma citri, which is also a plant pathogenic Mollicute, showed that disruption of an ATPase protein resulted in a lack of salivary gland penetration by S. citri (Garnier et al. 2001). In addition, disruption of an ABC transporter solute binding protein of S. citri resulted in a significant decrease in transmissibility of the organism to host plants but did not affect replication in the insect vector (Boutareaud et al. 2004). Insect vectors are only able to transmit the phytoplasma after their salivary glands have become infected, which results in a 2–6 week latent period between acquisition and transmission (Kirkpatrick 1989; Fletcher et al. 1998; Lee et al. 2000) (Fig. 1). Once an insect vector is infective, it will remain inoculative for life (Fletcher et al. 1998). After the insect latency period is complete, the insect is able to transmit the phytoplasma to a plant host by feeding on phloem tissue (Fletcher et al. 1998). The phytoplasma is transmitted from

the insect into the phloem through saliva, which is required for the lubrication of mouthparts during feeding (Garnier et al. 2001). Not all leafhoppers that ingest the phytoplasma during acquisition are able to transmit the phytoplasma (Blanche et al. 2003b). Transmission efficiency can be affected by the insect’s gender and life cycle (Beanland et al. 1999) and by temperature, age of the host plant and the nature of the host plant (Garcia-Salazar et al. 1991; Bosco et al. 1997; Palermo et al. 2001). There is a latent period between infection of a plant by an insect vector and symptom expression because the phytoplasmas multiply in the phloem of the plant host before they induce symptoms (Lee and Davis 1992) (Fig. 1). The phytoplasmas replicate in the phloem tissue of the ‘infected plant’ and can spread throughout the plant including the roots (Lee et al. 1998). Phytoplasmas can also be transmitted between host plants by cleft grafting, dodder (parasitic vine, Cuscuta spp.) or by vegetative propagation such as cuttings or rhizomes (Hibben and Wolanski 1970; Chiykowski 1988; Carraro et al. 1991; Gibb et al. 1995; Lee et al. 2000). Phytoplasmas have been detected in the embryo tissues of coconut palms with lethal yellowing disease which raises the issue of possible seed transmission of phytoplasmas (Cordova et al. 2003). However, seed transmission of phytoplasmas has not been demonstrated experimentally and the issue remains unresolved (Lee et al. 2000). Plants inoculated with a phytoplasma can exhibit floral symptoms such as virescence (green flowers), sterility, phyllody (floral organs develop leaf-like appearance) and gigantism (Lee and Davis 1992; Lee et al. 2000). Symptoms that affect other plant structures include profusion of auxillary buds (witches’ broom), stunting, dieback, yellowing, necrosis of phloem tissue, reduced foliar size (little leaf) and bunching at stem terminals (McCoy et al. 1989; Lee and Davis 1992; Lee et al. 2000). Symptom expression can be affected by environmental factors such as temperature, soil type, vitality and age of the plant at the time of infection which means there is no clear association between phytoplasma type and symptom development (Seemüller et al. 1998). Phytoplasma symptoms of mosaic, yellowing, chlorosis or mottling are thought to be due to a decrease in photosynthesis or disruption to carbohydrate translocation in the plant (Lepka et al. 1999). Alternatively, the phytoplasma may produce a metabolite that disrupts the integrity of the phloem tissue (Siddique et al. 1998; Guthrie et al. 2001). Symptoms of witches’ broom, little leaf and shortened internodes are thought to reflect a hormone imbalance in the diseased plants (Kuske and Kirkpatrick 1992). There are currently only a limited number of disease management strategies available to control the spread of phytoplasma disease. Generally, plants exhibiting phytoplasma-related symptoms are removed, which may be ineffective if weeds or native plants in the surrounding


area are reservoirs for the phytoplasmas associated with the disease in the crop (Angelini et al. 2004). Alternative approaches to control phytoplasma diseases are the use of netting to exclude vectors or white washing to deter insect feeding (Liu et al. 1996; Elder et al. 2002). The application of systematic insecticide to the area surrounding lucerne seed stands was found to reduce the migration of the insect vector, O. argentatus into the crop area (Pilkington et al. 2004a). Plantibody technology has been used to develop tobacco plants that express antibodies that are specific for the stolbur phytoplasma (Le Gall et al. 1998). Tobacco plants expressing the stolbur phytoplasma-specific antibodies showed resistance to this phytoplasma when grafted with diseased tobacco material, which suggests that plantibodyderived disease resistant cultivars may be an option to control these fastidious organisms (Le Gall et al. 1998). Economics and threat Phytoplasmas are associated with plant diseases that contribute to important crop losses worldwide (Lee et al. 2000). In Europe and North America, phytoplasmas associated with stone fruit diseases contribute to significant decreases in pear and peach yield (Lee et al. 2000). Coconut lethal yellowing is a devastating disease occurring in the Caribbean, Central and South America, Mexico and areas of Africa (Mpunami et al. 1999). Outbreaks of this disease have destroyed entire plantations in these regions (Mpunami et al. 1999). In Asia, rice production is limited by rice yellow dwarf disease while legume crop yields in this area are affected by peanut witches’ broom, sesame phyllody and soybean phyllody diseases (Lee et al. 2000). Phytoplasma diseases such as pear decline, European stone fruit yellows, aster yellows, sugarcane white leaf and coconut lethal yellowing could be devastating if they were introduced to other regions. To reduce this threat, movement of plant material from diseased areas is highly regulated. However, new phytoplasma disease associations are still reported worldwide on a regular basis so phytoplasmas may be moved between regions in plant material that is not recognised as being a phytoplasma host. Furthermore, diseased material can be unintentionally moved between regions in asymptomatic plants because of the lag time between inoculation of a plant and symptom expression. Phytoplasma disease outbreaks can occur locally from ‘infected’ plants adjacent to the crop. Alternatively, the newly introduced plant species may be a preferred feeding host for the insect vector and may also be susceptible to the phytoplasma (Tran-Nguyen et al. 2000; Padovan and Gibb 2001; Streten et al. 2005b). History of phytoplasma research—Australia Phytoplasma-type symptoms were first recorded in Australia in the early 1900s as tomato big bud (TBB) disease (Cobb 1902). In 1933, Samuel et al. reported that a


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virus was possibly associated with TBB disease. The diseases, lucerne witches’ broom (Edwards 1935; Helson 1951) and legume little leaf (Hutton and Grylls 1956) that exhibit symptoms typical of phytoplasma inoculation were also thought to be associated with a virus. In Australia, phytoplasmas were first observed in the phloem of periwinkle (Catharanthus roseus) and tobacco (Nicotiana glutinosa) plants exhibiting phyllody (Bowyer et al. 1969). Between 1970 and 1990, diagnosis of possible phytoplasma-associated diseases was based on electron-microscopy and transmission experiments (Bowyer and Atherton 1970, 1971; Bowyer 1974; Greber and Gowanlock 1979). During this time, a range of Australian plant diseases such as little leaf of French bean (Phaseolus vulagris) (Bowyer and Atherton 1970), dodder (Cuscuta australia) little leaf (Bowyer and Atherton 1970), strawberry lethal yellows (Greber and Gowanlock 1979), papaya yellow crinkle (Greber 1966), sweet potato little leaf (Gowanlock et al. 1976) and tomato big bud (Bowyer et al. 1969) were identified as being associated with phytoplasmas. In 1995, the association of a phytoplasma with tomato big bud disease was confirmed by molecular analysis (Gibb et al. 1995). Restriction fragment length polymorphism (RFLP) and sequence analysis of the TBB phytoplasma 16S rRNA gene showed that this phytoplasma was a member of the faba bean phyllody (FBP-) group which is assigned to the phytoplasma 16Sr group, 16SrII (Gibb et al. 1995; Schneider et al. 1995; Davis et al. 1997b). The faba bean phyllody group also includes Asian phytoplasmas such as sesame phyllody, peanuts witches’ broom and Chinese pigeon pea (Schneider et al. 1995; Seemüller et al. 1998; IRPCM Phytoplasma/Spiroplasma Working Team— Phytoplasma taxonomy group 2004). Due to the phylogenetic grouping of the TBB phytoplasma, it has been suggested that this phytoplasma may have originated from the closely related Asian phytoplasmas (Schneider et al. 1995; Davis et al. 1997b). Members of the phytoplasma 16SrII group have been designated ‘Candidatus Phytoplasma aurantifolia’ (Zreik et al. 1995). The sweet potato little leaf (SPLL) phytoplasma, which is closely related to the TBB phytoplasma, was transmitted by grafting from naturally infected sweet potato (Ipomoea batatas) plants collected near Darwin (Northern Territory, Australia) to periwinkle plants (Gibb et al. 1995). RFLP analysis of the 16S rRNA gene of the SPLL phytoplasma isolated from periwinkle showed slight variation in AluI and RsaI patterns compared with the TBB and SPLL phytoplasmas (Schneider et al. 1999a; Padovan et al. 2000a). The SPLL phytoplasma isolate in periwinkle was designated sweet potato little leaf strain V4 (SPLL-V4) phytoplasma to distinguish it from field samples of the SPLL phytoplasma (Padovan et al. 2000a). The TBB and SPLL-V4 chromosomes were isolated and subjected to RFLP analysis which demonstrated that genomic diversity existed between

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these phytoplasmas (Davis et al. 1997b; Schneider et al. 1999a; Padovan et al. 2000a). The SPLL-V4 phytoplasma is also associated with a range of plant diseases in Australia (Schneider et al. 1999b; Davis et al. 2003). The TBB and

SPLL-V4 phytoplasmas are highly successful in Australia because they are associated with a diverse range of host plant species (Tables 2 and 3) (Schneider et al. 1999b; Davis et al. 2003).

Table 2. Plant hosts and symptoms associated with the tomato big bud phytoplasma in Australia Genus species

Symptoms/disease

Citation

Alysicarpus rugosus Apium graveolens Arachis hypogea Boeharvia sp. Brugmansia candida Capsicum annuum Carica papaya

Witches’ broom Stunting, chlorosis, reddening Witches’ broom Little leaf, virescence Little leaf Little leaf and phyllody Yellow crinkle, mosaic

Catharanthus roseus Cenchrus ciliaris Cichorum intybus Citrus paradise Cleome viscose Crotalaria sp. (goreensis, novaehollandiae) Cucurbita maxima Cynodon dactylon Dactylorhiza majalis Daucus carota Emilia sonchifolia Eragrostis falcata Eriachne obtusa Euphorbia milli Gerbera sp. Goodenia sp. Guizotia abyssinica Lactuca sativa Lycopersicon esculentum Macroptilium sp. (bracteatum, atropurpureum, lathyroides) Mucuna pruriens Passiflora sp. Phlox sp. Physalis minima

Phyllody Bunchy shoots Little leaf and phyllody Dieback Little leaf Witches’ broom

Davis et al. (1997b) Tran-Nguyen et al. (2003) Davis et al. (1997b) Padovan and Gibb (2001) Davis et al. (1997b) Tran-Nguyen et al. (2003) De La Rue et al. (1999), Padovan and Gibb (2001) Davis et al. (1997b) Tran-Nguyen et al. (2000) Tran-Nguyen et al. (2003) Davis et al. (1997b) Davis et al. (2003) Davis et al. (1997b)

Little leaf Yellow leaves Virescence and stunting Hairy root Phyllody Grassy shoots Grassy shoots Little leaf Phyllody Little leaf Little leaf Phyllody Big bud Little leaf, witches’ broom

Davis et al. (1997b) Tran-Nguyen et al. (2000) Gowanlock et al. (1998) Gibb et al. (2003a) Schneider et al. (1999b) Tran-Nguyen et al. (2000) Tran-Nguyen et al. (2000) Davis et al. (1997b) Davis et al. (1997b) Schneider et al. (1999b) Davis et al. (1997b) Davis et al. (1997b) Davis et al. (1997b) Schneider et al. (1999b)

Witches’ broom Little leaf Phyllody Big bud, little leaf

Ptilotus distans Rhynchosia minima Saccharum sp. Sarochilus hartmanii Sesamum indicum

Phyllody Witches’ broom Yellow leaves, streaks Virescence Floral dieback

Sida cordifolia Solanum melongeria Stylosanthes scabra

Little leaf Little leaf, phyllody, big bud Witches’ broom

Trifolium repens Vigna sp. (lanceolata, luteolam trilobata, ungicillata) Vitis vinifera

Phyllody Witches’ broom

Zinnia elegans

Phyllody

Davis et al. (1997b) Schneider et al. (1999b) Davis et al. (1997b) Davis et al. (1997b), Padovan and Gibb (2001) Schneider et al. (1999b) Davis et al. (1997b) Tran-Nguyen et al. (2000) Davis et al. (1997b) Davis et al. (1997b), Wilson et al. (2001) Davis et al. (1997b) Davis et al. (1997b) Schneider et al. (1999b), De La Rue et al. (2001) Davis et al. (1997b) Davis et al. (1997b), Padovan and Gibb (2001) Constable et al. (1998, 2002, 2003), Gibb et al. (1999) Davis et al. (2003)

Yellows, late season leaf curl



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Table 3. Plant hosts and symptoms associated with the sweet potato little strain V4 phytoplasma in Australia Genus species

Symptoms/disease

Citation

Aeschynomene americana Aeschynomene indica Alysicarpus vaginalis

Little leaf Little leaf Little leaf, witches’ broom

Aphyllodium sp. Arachis sp. (hypogea, pintoii)

Phyllody Little leaf, phyllody

Cajanus marmoralus Carica papaya Catharanthus roseus Centrosema pascuorum Citrus sp. Cleome viscose

Little leaf Yellow crinkle, mosaic Virescence, witches’ broom Little leaf — Little leaf

Crotalaria sp. (brevis, crispate, goreensis, pallida) Cucurbita maxima Cyanthilium cinereum Cyanthillium cinereum Desmodium sp. (intortum, triflorum) Emilia sonchifolia Indigofera sp. (colutea, linifolia, hirsuta) Ipomoea sp. (pescaprae, plebeian) Macroptilium gracile Medicago sativa Mitracarpus hirtus Nicotiana tabacum Pachyrhizus erosus Physalis minima Rhynchosia minima

Phyllody, little leaf, witches’ broom Fasciation Witches’ broom Witches’ broom Little leaf Phyllody Little leaf, phyllody, proliferation Little leaf Little leaf Little leaf Little leaf Big bud Little leaf Big bud Witches’ broom, little leaf

Wilson et al. (2001) Padovan and Gibb (2001) Wilson et al. (2001), Davis et al. (2003) Padovan and Gibb (2001) Padovan and Gibb (2001), Wilson et al. (2001) Padovan and Gibb (2001) De La Rue et al. (1999) Davis et al. (1997b, 2003) Wilson et al. (2001) Davis et al. (2003) Padovan and Gibb (2001), Davis et al. (2003) Padovan and Gibb (2001), Davis et al. (2003) Padovan and Gibb (2001) Schneider et al. (1999b) Davis et al. (2003) Padovan and Gibb (2001), Davis et al. (2003) Davis et al. (2003) Padovan and Gibb (2001)

Senna obtusifolia Sesamum indicum Stylosanthes sp. (scabra, hamata)

Little leaf Floral dieback, phyllody Little leaf

Tridax procumbens

Little leaf

Most phytoplasmas found in Australia are assigned to the faba bean phyllody group, but phytoplasmas associated with some economically important diseases such as Australian grapevine yellows (AGY) are assigned to the phytoplasma 16Sr group (Padovan et al. 1995, 1996; Schneider et al. 1999b) (Tables 2, 3 and 4). A phytoplasma association with AGY disease was confirmed by PCR using primers specific for the phytoplasma 16S rRNA (Padovan et al. 1995). RFLP and nucleotide sequence analysis of the AGY phytoplasma 16S rRNA gene showed that this phytoplasma is most closely related to German grapevine yellows and stolbur phytoplasmas so the AGY phytoplasma was assigned to the aster yellows (16SrI) group (Padovan et al. 1995, 1996). However, the AGY phytoplasma can be differentiated from other members of the 16SrI group based on 16S rRNA gene

Davis et al. (2003) Padovan and Gibb (2001) Wilson et al. (2001) Wilson et al. (2001) Padovan and Gibb (2001) Davis et al. (2003) Padovan and Gibb (2001) Wilson et al. (2001), Davis et al. (2003) Padovan and Gibb (2001) Wilson et al. (2001) De La Rue et al. (2001), Padovan and Gibb (2001) Padovan and Gibb (2001)

RFLP banding patterns when digested with MseI and AluI (Padovan et al. 1995, 1996) so Davis and Sinclair (1998) moved the AGY phytoplasma from the 16SrI group into the stolbur group (16SrXII) and designated it subgroup B. Davis et al. (1997a) assigned the AGY phytoplasma the candidate species name Candidatus Phytoplasma australiense based on the identification of signature sequences in its 16S rRNA gene and RFLP data. Liefting et al. (1998) proposed that the Australian papaya dieback (PDB) phytoplasma should also be designated Ca. P. australiense because its 16S rRNA gene has the AGY phytoplasma signature sequences. In Australia, the common brown leafhopper, Orosius argentatus, was identified as the vector of the TBB phytoplasma in 1943 (Hill 1943) and this phytoplasma– insect association has since been confirmed by additional

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Table 4. Phytoplasmas and their plant host species in Australia excluding tomato big bud and sweet potato little leaf strain V4 Phytoplasma 16Sr group

Phytoplasma

Genus species

Symptoms

Citations

16SrI

Buckland valley grapevine yellows (BVGY)

Vitis vinifera

Yellows

Constable et al. (1998, 2002), Gibb et al. (1999)

16SrII

Australian lucerne yellows (ALuY) Bonamia pannosa (BoLL) Cactus witches’ broom (CaWB) Cocky apple witches’ broom (CAWB) Pigeon pea little leaf (PLL)

Medicago sativa

Yellows

Pilkington et al. (2003)

Bonamia pannosa

Little leaf

Carica papaya

Yellow crinkle

Planchonia careya

Witches’ broom

Schneider et al. (1999b), Padovan and Gibb (2001) De La Rue et al. (1999), Padovan and Gibb (2001) Davis et al. (2003)

Arachis sp. (hypogaea, pintoii) Catharanthus roseus Crotalaria sp. (goreensis, spectabilis) Desmodium triflorum Indigofera sp. (colutea, linifolia, hirsuta) Macroptilium bracteatum Pterocaulon sp. Sesuvium portulacastrum Stylosanthes scabra

Little leaf, phyllody Witches’ broom Phyllody, proliferation Little leaf Stunting, phyllody, little leaf Little leaf Yellowing, rosette Little leaf Witches’ broom, little leaf Little leaf, phyllody Little leaf Yellow crinkle Little leaf Decline Bunching, little leaf Yellow streaks Little leaf Little leaf Little leaf, proliferation Yellow crinkle

Sweet potato little leaf (SPLL)

Waltheria little leaf (WaLL)

Vigna radiata Cajanus cajan Carica papaya Ipomoea batatas Pyrus communis Mitracarpus hirtus Saccharum sp. Spermacocci sp. Waltheria indica Sida sp. Carica papaya

Schneider et al. (1999b), Wilson et al. (2001) Davis et al. (2003) Schneider et al. (1999b), Davis et al. (2003) Davis et al. (2003) Padovan and Gibb (2001) Schneider et al. (1999b) Wilson et al. (2001) Davis et al. (2003) Schneider et al. (1999b), De La Rue et al. (2001) Wilson et al. (2001) Davis et al. (1997b) Liu et al. (1996) Gibb et al. (1995) Schneider and Gibb (1997) Wilson et al. (2001) Tran-Nguyen et al. (2000) Schneider et al. (1999b) Schneider et al. (1999b) Padovan and Gibb (2001) Padovan and Gibb (2001)

16SrIII

Poinsette branching (PoiBI) Weeping tea tree witches’ broom (WTWB)

Euphorbia pulcherrima Melaleuca sp. (cajuputi, leucadendra)

Branching Witches’ broom

Schneider et al. (1999b) Davis et al. (2003)

16SrX

Allocasuarina yellows (AlloY)

Allocasuarina muelleriana

Witches’ broom

Gibb et al. (2003b)

16SrXII

Candidatus Phytoplasma australiense

Carica papaya

Dieback

Catharanthus roseus Cucurbita maxima Fragaria × ananassa Gomphocarpus physocarpus Phaseolus vulgaris Phaseolus aureus

Phyllody, witches’ broom Yellow leaf curl Green petal, yellows Witches’ broom Witches’ broom Witches’ broom

Paulownia sp.

Stunted growth, interveinal chlorosis Yellows, late season leaf curl, restricted growth

Liu et al. (1996), Gibb et al. (1998) Davis et al. (2003) Streten et al. (2005a) Padovan et al. (1998, 2000b) Streten et al. (2005b) Schneider et al. (1999b) Davis et al. (1997b), Streten and Gibb (2005) Bayliss et al. (2005)

Vitis vinifera

Davis et al. (1997b), Gibb et al. (1999), Constable et al. (2003) (Continued next page)



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Table 4. (continued) Phytoplasma 16Sr group

Phytoplasma

Genus species

Symptoms

Citations

Cenhrus bunchy shoot (CBS)

Cenchrus setiger

Bunchy shoot

Tran-Nguyen et al. (2000)

16SrXIV

Cynodon white leaf (CWL)

Cynodon dactylon

White leaf

Dactylocterium aegyptiuma

White leaf

Schneider et al. (1999b), Tran-Nguyen et al. (2000) Blanche et al. (2003a)

No group assigned

Galactia little leaf (GaLL) Sorghum bunchy shoot (SBS) Sorghum grassy shoot (SGS)

Galactia tenuiflora

Little leaf

Sorghum stipoideum

Grassy shoot

Chloris inflate Dactylocterium sp. (aegyptiuma, radulans) Sorghum stipoideum Whiteochloa sp. (bicilata, cymbiformis, capillipes) Arachis pintoii Bonamia pannosa Carica papaya Indigofera linifolia Saccharum sp. Sesuvium portulacastrum Stylosanthes scabra

Creamy leaves White leaf, grassy shoot, stunting Grassy shoot, white leaf White leaf, grassy shoot Little leaf Little leaf Yellow crinkle Phyllody, little leaf Yellow streaks Little leaf Little leaf

Saccharum sp.

Yellow streaks

Stylosanthes scabra Tridax procumbens Vigna lanceolata

Little leaf Phyllody Little leaf

Stylosanthes little leaf (StLL)

Sugarcane phytoplasma (SCP) Vigna little leaf (ViLL)

vector studies (Hutton and Grylls 1956; Greber 1966). Within Australia, no insect vectors of Ca. P. australiense have been identified, but in New Zealand the planthopper, Oliarus atkinsoni transmits this phytoplasma (Boyce and Newhook 1953; Liefting et al. 1997). O. atkinsoni is a monophagous species that feeds on Phormium sp. and is essentially limited to New Zealand (Boyce and Newhook 1953; Liefting et al. 1997; Andersen et al. 2001) so it is unlikely to transmit Ca. P. australiense in Australia. A survey of phloem feeding insects in papaya plantations in Queensland where dieback, yellow crinkle and mosaic diseases occur, found seven different planthopper species and 13 different leafhopper species were present, but these insect species were negative for phytoplasma when screened by PCR (Elder et al. 2002). A survey of papaya plantations in the Northern Territory detected the Stylosanthes little leaf and sweet potato little leaf strain V4 phytoplasmas in the bodies of Orosius sp. and the Vigna little leaf phytoplasma in Austroagallia torrida and Batracomorphus sp. when assayed by PCR

Schneider et al. (1999b), Padovan and Gibb (2001) Tran-Nguyen et al. (2000) Blanche et al. (2003a) Schneider et al. (1999b), Blanche et al. (2003a) Schneider et al. (1999b), Blanche et al. (2003a) Blanche et al. (2003a) Schneider et al. (1999b) Padovan and Gibb (2001) Padovan and Gibb (2001) Padovan and Gibb (2001) Tran-Nguyen et al. (2000) Davis et al. (2003) Schneider et al. (1999b), De La Rue et al. (2001) Tran-Nguyen et al. (2000) De La Rue et al. (2001) Padovan and Gibb (2001) Schneider et al. (1999b), Padovan and Gibb (2001)

(Padovan and Gibb 2001). Pilkington et al. (2004a) reported that A. torrida and B. angustatus are also possible candidate vectors for the Australian lucerne yellows (ALuY) phytoplasma based on a correlation between the occurrence of symptomatic plants and leafhopper populations. In transmission trials with A. torrida collected from lucerne seed crops, five lucerne plants exhibited possible phytoplasmarelated symptoms while in insect trials with B. angustatus, all plants were non symptomatic (Pilkington et al. 2004b). PCR and RFLP analysis of the symptomatic lucerne plants showed that a single plant exhibiting stunting and dieback symptoms was positive for the TBB phytoplasma which provides experimental evidence that A. torrida is a vector for this phytoplasma (Pilkington et al. 2004b). Pilkington et al. (2004b) reported O. argentatus as a possible vector for the ALuY phytoplasma. This leafhopper was identified as a vector of the ALuY phytoplasma based on insect transmission trials and electron microscopy of phloem tissue, but the phytoplasma type present in the symptomatic

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plant could not be characterised by RFLP analysis (Pilkington et al. 2004b). The leafhopper, O. lotophagorum subsp. ryukyuensis, was identified as a vector for the phytoplasmas associated with little leaf and phyllody disease of bellvine (Ipomoea plebeian) plants in south-east Qld by insect transmission trials and electron microscopy (Behncken 1984). Tropical and sub-tropical crops and phytoplasmas in Australia Tropical and sub-tropical Australia The Australian continent is divided into three latitudinal climate zones; these are tropical, sub-tropical and temperate (Linacre and Hobbs 1977). The tropical zone covers Northern Australia (5–20◦ S) which includes towns such as Darwin (NT), Katherine (NT), Kununurra (WA), Cairns (north Qld) and Rockhampton (south central Qld) (Linacre and Hobbs 1977; Fox 1999; http://www.bom.gov.au). The tropical region of Australia receives intense rainfall from monsoons or low depressions in summer, also know colloquially as ‘the wet’ (November–March) (Linacre and Hobbs 1977; Fox 1999). The mean maximum temperature for January (wet season) is 30◦ C. From April to October (winter or ‘the dry’) the tropics are in drought, temperatures range from 19–33◦ C, and trade winds blow from the south-east (Linacre and Hobbs 1977). The sub-tropical zone in Australia (20–30◦ S) includes towns such as Alice Springs (NT), Brisbane (south east Qld) and Nambour (south east Qld) (Linacre and Hobbs 1977; http://www.bom.gov.au, verified 2 February 2006). The sub-tropics are considered the arid region of Australia, rainfall in this region is variable and the average rainfall is