Keywords

6.1 Introduction

Doi and coworkers in 1967 discovered by electron microscopy observations pleomorphic, wall-less bacteria in the phloem of many plant species affected by yellows-type diseases previously believed to be caused by viruses (Kunkel 1926, 1931, 1955; Nasu et al. 1967; Mc Coy et al. 1989; Lee and Davis 1992; Maramorosch 2011). These microorganisms named mycoplasma-like (MLO) were later renamed phytoplasmas since they were shown to be clearly differentiable from mycoplasmas, recognized pathogens for human and animals since long time (Nocard and Roux 1898), for their ribosomal RNA gene (Lim and Sears 1989) and are now classified based on molecular analyses on the 16S ribosomal gene at the level of the ‘Candidatus’ genus. The disappearance of symptoms after antibiotic (i.e. tetracycline) treatments provided additional evidence to support their pathogenic role in the diseased plants (Ishiie et al. 1967). After the discovery of MLO, the plant pathologists took up the challenge to culture these organisms in growth media. In the early 1970s, reports on MLO cultivation were published (Lombardo and Pignattelli 1970; Lin et al. 1970; Giannotti and Vago 1971; Ghosh et al. 1975; Skripal et al. 1984), but none of the described methodology proved to be repeatable, and scientists start to consider phytoplasmas as unculturable microorganisms (Maramorosch 2011). Trials were carried out using media able to support the mycoplasma or the spiroplasma growth (Skripal and Malinivskaya 1984; A. Bertaccini, 1990, unpublished; Poghosyan and Lebsky 2004) until phylogenetic data on mollicutes (Gasparich et al. 2004) clearly linked the phytoplasmas to one of the mycoplasma groups and mycoplasma-based media were tested for phytoplasma cultivation (Fig. 6.1) with the successful isolation of 16SrXII-A phytoplasmas from micropropagated periwinkle shoots (Bertaccini et al. 1992, 2010).

Fig. 6.1
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From left: scheme used for first phytoplasma isolation from micropropagated periwinkles in collection and isolation tubes containing infected plant materials; the tube labeled C-contains healthy plant materials

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By continuation of these trials, phytoplasma cultivation in artificial media was achieved (Contaldo et al. 2012) using diverse phytoplasmas in micropropagated infected periwinkle shoots such as Chrysanthemum yellows (strain CY-TO from Italy, 16SrI-B), tomato big bud (strain TBB from Australia, 16SrII-D) and ‘‘stolbur’’ (strains CH-1 from Italian grapevine and STOL, from Serbian pepper). These results were confirmed by the isolation soon after of the potato witches’ broom (strain PWB from the USA, 16SrVI-A), lime witches’ broom (strain WBDL from Oman, 16SrII-B) and Picris echioides yellows (strain PEY from Italy, 16SrIX-C) phytoplasmas (Contaldo et al. 2013, 2014a). More recently, by using the same media, phytoplasmas were isolated and grown from cassava infected by the frog skin disease associated with 16SrIII-L strain employing fragments of roots, petioles, stems, leaves and embryos as a source for phytoplasma isolation (Alvarez et al. 2009, 2010; Rao et al. 2018).

6.2 Isolation Media Development

Attempts to cultivate phytoplasmas have been performed using artificial media similar to those used for spiroplasma growth (Davis et al. 1972; Saglio et al. 1973; Müller et al. 1975; Elmendorf 1977; Whitcomb and Tully 1975; Jones et al. 1977), but the experiments were not repeatable, and the results were therefore not accepted by the scientific community. To explain the difficulties in cultivating phytoplasmas, two hypotheses have been formulated: the developed media lack essential elements and phytoplasmas are host cell-dependent. The first growth in artificial media was obtained when infected micropropagated periwinkle shoots were used as source and the isolation was carried out on available commercial media with composition not disclosed by the provider (Mycoplasma Experience Ltd) (Contaldo et al. 2012). In order to achieve the cultivation from naturally diseased plant materials, media supporting the growth from different plants sources and with flexible and modifiable composition were specifically designed (Contaldo et al. 2016a). These complex media (CB) are based on TSB (Oxoid, UK) pH 7.3 ± 0.2 which essentially contains tryptone and soy peptone, and enriched with a supplement containing 20 ml of sterile horse serum (Oxoid, SR0035), and 25 μg/ml of ampicillin (Sigma, A9393) and 50 μg/ml of nystatin (Sigma, N6261) both 0.22 μm filter sterilized, 10 ml of autoclaved yeast extract (25% w/v) and 0.005% of phenol red for each 80 ml of medium. These media were used to support the isolation and growth of phytoplasmas from infected field-collected grapevine samples. Comparative trials showed that the new developed CB medium could support the phytoplasma growth in the same manner as the previous Piv media. In spite of the relatively long time required for incubation in liquid medium (from 2 to 15 days), colony growth on agar usually occurs within 2–5 days (Fig. 6.2) as for the majority of cultured bacteria.

Fig. 6.2
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Phytoplasma colonies photographed under binocular microscope (40×)

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The composition of solid medium, CBs, was mainly TSB, NaCl 20 g/l, agar No. 3 12 g/l (Oxoid, LP0013), ampicillin and nystatin as before. One of the key points that was shown to be important within phytoplasma isolation from plant sources was the optimization of the growing conditions, by the use of plate incubation in a specific 2.5 l anaerobic jar (Oxoid, AG0025) in a microaerophilic atmosphere using CampyGen sachets (Oxoid, CN0025) (Contaldo et al. 2014b, c, 2016a). The results obtained from phytoplasma isolation and cultivation from field-infected grapevine samples were confirmed by the phytoplasma isolation from coconut palms infected by Côte d’Ivoire lethal yellowing (CILY) (Fig. 6.3) (Contaldo et al. 2019). The samples employed for the phytoplasma isolation and culturing were representative of the various stages of the disease (Arocha Rosete et al. 2017), and leaves, inflorescences and trunk boring were the tissues employed for the isolation.

Fig. 6.3
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On the left CILY symptoms on coconut palms at different CILY disease stages in Côte d’Ivoire (kindly provided by Y. Arocha-Rosete) and on the right grapevine leaves infected with grapevine yellows phytoplasmas (GY) used for phytoplasma isolation: top a red grapevine variety and bottom a white variety

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While the leaves and the inflorescences were used as described (Contaldo et al. 2016a), a change in the isolation procedure was employed for the trunk boring samples. In fact, approximately 100 mg from each plant were placed in two separate sterile mortars and mixed with 1 ml of CBl using a sterile pestle. Immediately after, 100 μl of the mixtures were transferred into monovette tubes and incubated (Contaldo et al. 2019).

The use of commercial TSB in liquid and solid media formulation for phytoplasma isolation and subsequent culture supports also the growth of bacterial endophytes present in the plant source materials (Fig. 6.4). Different combinations of antibiotics, carbon sources and NaCl concentration were therefore evaluated by visual observation of the degree of turbidity of the liquid media, due to the presence of contaminating microorganisms and/or symbionts inside the isolation tissue, and by the ability to form colonies in agar (Fig. 6.5) (Contaldo et al. 2018a).

Fig. 6.4
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From the left endophytes and phytoplasma colonies in a plate and (right) photographed under a bifocal optical microscope (40 X) two days after plating

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Fig. 6.5
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Close-up: two Petri dishes on the left with colonies treated with tetracycline and on the right treated with penicillin

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The colony purification was performed by gentle filtration of the entire liquid culture through 0.8 μm membrane filters (Whatman, Maidstone, UK) to avoid plugging, followed by 10−3/10−4 serial dilutions in CBl. Both the undiluted filtrate and the filtrate dilutions were cultured on solid CB plates and incubated at 25 ± 1 °C. This process, including the filtration, is repeated three times from single descending colonies (Fig. 6.6).

Fig. 6.6
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On the left: Petri dish containing phytoplasma colonies derived from the 10−3 liquid media dilutions showing the purification by streaking onto plate. On the right: phytoplasma colonies after the purification procedure photographed with binocular microscope

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6.3 Phytoplasma Identification in Liquid and Solid Media

One of the main challenges with phytoplasma cultivation is to define a rapid, repeatable and reliable detection method from both solid and liquid cultures. The DNAs extracted from both media were amplifiable on 16Sr gene only after nested PCR with universal primers and only scattered amplification was obtained on other nonribosomal genes.

This was the case of amplicons obtained on ef-Tu gene on aster yellows (AY) and ‘‘stolbur’’ (STOL) isolates from periwinkle shoots (Contaldo et al. 2012) and of STOL colonies grown on agar media after isolation from field-collected grapevine that tested positive on stamp gene (Fig. 6.7) (Contaldo et al. 2016b). The RFLP profiles and their sequences obtained after direct sequencing in both directions with primers used for amplification are however useful to confirm the phytoplasma presence and identity (Fig. 6.8) (Contaldo et al. 2012, 2015a, b). The difficulty in amplifying nonribosomal genes from these isolates is probably due to both low DNA concentration (number of cells) and the presence of mixed bacterial and/or phytoplasma population in the media.

Fig. 6.7
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From the left ‘‘stolbur’’ phytoplasma colonies in agar plate two days after inoculation under bifocal microscope (40 X) and left polyacrylamide (6.7%) gel showing RFLP profiles of the stamp gene amplicons of the same colonies digested with Tru1I. M, Marker DNA phiX174 HindIII digested

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Fig. 6.8
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Phylogenetic tree showing the evolutionary relationships of reference phytoplasmas and phytoplasma strains isolated from coconut palms infected by CILY disease (in green) using the neighbour-joining method in MEGA6

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In the cassava frog skin isolation, the phytoplasma identity was verified using a quantitative PCR technique specific for the isolated phytoplasma (‘Candidatus Phytoplasma pruni’-related). Furthermore, the pathogenicity of five isolates containing phytoplasma DNAs was proved using stem injection in in vitro cassava shoots: severe CFSD and typical root symptoms were observed 6 months after inoculation (Alvarez et al. 2017). A preliminary qPCR analysis using universal primers designed on the 16Sr gene (Saccardo et al. 2012) was recently set (Contaldo et al. 2019) to verify the phytoplasma presence and titre in two culture suspensions obtained from CILY-infected coconut palms; at the inoculation time (T0) and after 18 h (T18), the methodology was able to demonstrate the increase of the phytoplasma titre.

6.4 Biological and Nutritional Properties of Selected Phytoplasma Strains

Some biological and nutritional properties of ‘Ca. P. asteris’ strains isolated from grapevine were determined. They were not surviving with sucrose as source of carbon and were very well differentiable from the Acholeplasma laidlawii strain used as control for the arginine hydrolysis ability (Contaldo et al. 2018a).

The same results were confirmed in ‘Ca. P. solani’ (16SrXII-A) and ‘Ca. P. palmicola’ (16SrXXII-B) isolates from coconut tissues. For these two latter strains, the antibiotic susceptibility was also evaluated (Fig. 6.9). The two strains studied, indeed, revealed the maximum susceptibility to tobramycin, followed by polymyxin and tetracycline; an intermediate susceptibility to 5-fluorouracil was also observed, but they resulted completely resistant to cephalexin hydrate and rifampicin (Contaldo et al. 2019).

Fig. 6.9
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Phytoplasma colonies treated with tetracycline with the standard disc diffusion method: on the left inhibition halo produced by the antibiotic on the colonies and on the right colonies inside and outside the inhibition halo photographed under binocular microscope (40 X)

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The biochemical characterization of the ‘Ca. P asteris’, ‘Ca. P. solani’, and ‘Ca. P. palmicola’ isolates revealed that they shared metabolic features with Mycoplasma species group III (glucose positive/arginine positive), which are represented by several mycoplasma species (i.e. Mycoplasma fermentans) (Brown et al. 2007; Freundt et al. 1979).

6.5 Mixed Phytoplasma Infection

The isolation of phytoplasmas from naturally infected grapevine and coconut plants highlighted how starting from samples in which one phytoplasma was detected by PCR could provide colonies in which different phytoplasmas are detected (Contaldo et al. 2018b, 2019). These results suggest that the medium employed is not phytoplasma specific and support the growth of phytoplasmas that are present in the endobiome of the plants at concentration that are below the routine threshold of molecular PCR detection.

The comparison between the phytoplasma subgroups found in the plant material prior to culture, and those found in the liquid and solid media, indicates the frequent presence of phytoplasmas in the tissues used for isolation that are not the same as those isolated. These somewhat contradictory results on phytoplasma identification obtained from plant material, liquid and solid cultures, confirm the common presence of a mixed phytoplasma infection. While it is very likely that the prevalent strain in the plant material is the one determined by the molecular analyses in plants, the medium used in these isolation trials appears to favour phytoplasmas enclosed in specific ribosomal groups. Moreover, also the differences in the identity of the phytoplasma that were detected from testing the DNA extracts from the CB liquid cultures and plate colonies further indicate the presence of mixtures of phytoplasmas. The presence of multiple phytoplasmas in a single plant host may modulate the expression of disease symptoms, as it was suggested regarding the presence of multiple apple proliferation phytoplasma strains in apple trees or for other phytoplasma-associated diseases (Seemüller and Schneider 2007; Rid et al. 2016). On the other hand, the medium-dependent growth kinetics for ‘Ca. P. solani’ (16SrXII-A) and ‘Ca. P. palmicola’ (16SrXXII-B) isolates from coconut indicated their different performances in the used medium. This phenomenon was clearly evidenced by the longer growth timeframe for the 16SrXII-A isolate. The 16SrXXII-B phytoplasma corresponds to the prevalent subgroup associated with CILY in Côte d’Ivoire; however, this isolate was shown to multiply less efficiently than the other in the media used. The shorter survival time in the artificial medium for this isolate, may be associated with the presence of a limiting factor in the cell-free medium, clearly demonstrating that the various phytoplasma isolates exhibit different performances in the same growth medium. This is possibly related to different biological behaviours, and indicates that very likely diverse phytoplasmas possess different plant colonization abilities. Further biochemical comparative studies may lead to the identification of new chemicals or bio-compounds which may improve the media composition to support a better growth performance and survival rate of other phytoplasmas or phytoplasma strains.

6.6 Insect Transmissibility from Colonies

Trials were carried out to verify if the phytoplasmas detected in the colonies are able to be acquired by insects. Aster yellows colonies from grapevine (isolates Gl1is and Gl2is) were used to investigate the acquisition ability of the leafhopper Scaphoideus titanus Ball that was demonstrated to transmit aster yellows phytoplasma through the eggs (transovarial transmission) (Danielli et al. 1996; Alma et al. 1997) and is able to acquire and transmit this phytoplasmas to plants under controlled conditions (Alma et al. 2001) since in nature it transmits only “flavescence dorée” phytoplasmas (Mori et al. 2002). Preliminary results showed that the different S. titanus nymph stages that were feeding on solid medium with and without phytoplasma colonies showed a good capacity of survival with mean survival time (LT50) of 4.4 ± 0.13 and 5.5 ± 0.35 days for nymph instars and adults, respectively (Fig. 6.10).

Fig. 6.10
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Steps for the insect acquisition on plates containing phytoplasma colonies

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Insects fed for 24 hours on phytoplasma-inoculated solid medium showed a percentage of phytoplasma acquisition ranging from 55% to 80%. The presence of aster yellows (AY) in the insects was detected up to 15 days post acquisition. The 16S ribosomal gene of phytoplasma detected in leafhopper at 7 days post inoculation (AY-St1) was 99% identical to the ones detected in the culture used for insect feeding. The alignment of these amplicons with aster yellows strains OY-M (16SrI-B) and AY-WB (16SrI-A) shows the presence of four SNPs between isolate Gl-1is and strain AY-St1; two of these SNPs are in common between AY-St1 and strain AY-WB, while the other two SNPs are specific for strain AY-St1 (Fig. 6.11). The nested PCR amplification of 30 S. titanus specimens not used for acquisition trials and maintained on healthy grapevine resulted in 28 negative specimens and 2 positive to specific 16SrV group primers, “flavescence dorée” possible phytoplasmas (Angelini et al. 2018), indicating that no 16SrI infection was present in the S. titanus rearing. Some of the healthy grapevine plants employed for insect feeding during the experiment were tested after 10 months and resulted positive to ‘Ca. P. asteris’ presence.

Fig. 6.11
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Alignment of aster yellows strains: Gl-1is, GenBank Accession Number (Acc. No.) KP890829; AY-St1, Acc. No. KP890830; aster yellows strains OY-M (16SrI-B, Acc. No. NC_005303); and AY-WB (16SrI-A, Acc. No. NC_007716). The four SNPs are between isolate Gl-1is and strain AY-St1; two of these are in common between AY-St1 and AY-WB (red squares), while the other two are specific for strain AY-St1 (green squares)

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6.7 Viability of Seed Transmitted Phytoplasmas

Phytoplasma seed transmission is still a poorly investigated topic in spite of the growing evidences of its presence in several agricultural relevant species. In order to better understand the mechanisms behind it and to verify the viability of phytoplasmas in the progenies, phytoplasma isolation trials were conducted from plantlets derived from symptomatic and phytoplasma-infected corn seeds (Satta et al. 2016, 2019). Preliminary trials were carried out on nine seedlings, six of which, positive to aster yellows and “stolbur” phytoplasmas, were used as sources for isolation trials. From three plants the isolation allowed to detect aster yellows and “stolbur” phytoplasma DNAs after chloroform/phenol extraction from 1 ml of liquid medium from both isolation tubes and from tubes obtained after serial dilution. Further tests on 79 seedlings resulted in 17 of them positive for aster yellows and “stolbur” phytoplasmas. Isolation from three of these plants after 30 days from germination resulted positive for phytoplasma DNA in tubes deriving after 2–10 serial dilution in fresh medium. Reisolation carried out at 90 days from germination confirmed these results from one plant. Plating carried out together with DNA extraction from liquid medium produced colonies of different sizes and shapes. The same type of colonies was obtained from plating tubes maintained for 7 months at 25°C. Single colonies were picked and transferred in broth for purification steps at several times, and small colonies were obtained resulting to consistently contain aster yellows DNAs; moreover this type of colony growth was observed for at least three subsequent passages in liquid/solid media carried out every 5 days. These preliminary results are indicating the viability of phytoplasmas isolated from corn seedlings.

6.8 Conclusions

The phytoplasma cultivation was an important breakthrough in the study of their biology since, despite a reduced genome size in comparison to their ancestors, their retain an independent metabolism that allows them to survive outside the host species very likely with restricted ability to grow in artificial media. This versatility is a unique property among plant-inhabiting microbes cultured up to now, shared only with some animal- or plant-infecting viruses and with a few other microorganisms such as the causal agent of malaria. The phytoplasma isolation and biological characterization are relevant for disease management and containment measures to contrast their epidemics.