Scanning electron microscopy detection of phytoplasmas and other phloem limiting pathogens associated with emerging diseases of plants

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1 Scanning electron microscopy detection of phytoplasmas and other phloem limiting pathogens associated with emerging diseases of plants V. Lebsky and A. Poghosyan CIBNOR, S.C., Avenida Inst. Politecnico Nacional 195, Playa Palo de Santa Rita Sur, La Paz, BCS, 23096, Mexico Scanning electron microscopy (SEM) was applied to detect the phytoplasmas and other phloem inhabiting pathogens associated with some emerging diseases of agricultural crops. The original technique of specimens preparation was used to observe the internal structure of phloem tissue. Phytoplasma cells ranging from 400 to 2000 nm were observed in phloem tissue of all analyzed samples of different crops and wild plants with strong foliar malformations and other diverse symptoms of yellow type diseases. Some asymptomatic samples had low concentration of phytoplasmas. In phloem tissue of some samples of Solanaceae (tomato and pepper) along with phytoplasmas groups of geminated particles characteristics of geminiviruses (Geminiviridae) were detected, as well as some rod shaped bacteria. In some samples from diseased citrus species (Rutaceae) phytoplasmas were observed together with large (up to 2500 nm) rickettsia-like bacteria, very similar to those reported for Candidatus liberibacter. A high content of organic and inorganic crystals and starch granules were noted in diseased plants, a phenomenon supposedly related with pathogenic pathway. Keywords: SEM; pathogen detection; phloem tissue 1. Introduction Plant diseases caused by distinct pathogens create challenging problems in agriculture and pose real economic threats. Phytoplasmas, a phloem inhabiting, wall-less prokaryotes, are causal agents of more than 700 plant diseases, some of which with high economic and quarantine importance. [1]. Phytoplasmas as well as other vascular prokaryotes and some viruses produce somewhat similar yellow type symptoms in infected plants [2-5]. Some symptoms known to be associated with phytoplasmas may be the result of co-infection with distinct pathogens [2,3]. Such cases need in accurate and timely diagnosis by the set of diagnostic procedures to formulate early disease management strategies. In the state of Baja California Sur symptoms of yellow-type diseases were observed in different crops and wild plants during phytopathological surveys in the main agricultural areas during [6]. In some tomato, pepper and citrus plantings a high incidence of emerging diseases with symptoms of strong foliar malformations, crinkled leaf veins and other diverse symptoms suggested a possible mixed infection with phytoplasmas and other pathogens. [2, 6-9]. Disease indexing and scanning electron microscopy (SEM) technique were performed to analyze the pathogens in collected samples prior to confirm the results by molecular techniques. 2. Materials and Methods 2.1 Sample collection and grafting Samples of symptomatic and asymptomatic plants with yellow type symptoms were collected during field surveys in commercial plantings and backyard plots in the main agricultural areas in the state of BCS. With the aim to determine the presence of pathogen in various growth stages we used disease indexing in test-plants by grafting. 2.2 SEM analysis Samples from leaf midribs, leafstalks, young leaflets and flowers from symptomatic and asymptomatic plants were fixed in 2.5% solution of glutaraldehide with sodium cacodilate buffer 0.2 M (ph ) at 4 C during hours. The specimens were washed in the same buffer (20 min) and then dehydrated in solutions of alcohols at 30, 50, 70, 96 y 100%, followed by absolute acetone or hexamethyldisilazane, for 20 min in each solution. After the dehydration, the specimens were dried in the carbon dioxide in critical point dryer (Samdri PVT-3B), attached to SEM stubs and coated with palladium in an ion sputter (DESK II, Denton Vacuum), for 40 seconds. Before coating with palladium, the samples were incised using razor to observe the internal parts of plant tissues.. Prepared specimens were examined in a SEM (S-3000N Hitachi) at 5-20 kv. 78

2 3. Results and discussion 3.1 SEM analysis of tomato and pepper Phytoplasma cells were detected in phloem tissue of both field and greenhouse indexed samples of diseased tomato and pepper (Figs.1, 3). They looked like rounded particles, ranging from 400 to 2000 nm, separated or clusters. Phytoplasmas were revealed during all the vegetation period in distinct plant organs: leaves, leafstalks, roots, floral parts, but in different concentration in distinct growth stages. Some phytoplasma cells observed near and within sieve pores of sieve elements indicating their mode of transmission through the phloem tissue (Figs. 1b, c). Some rod-shaped bacteria with average sizes 1000 x 250 nm observed in some phloem tissues in which phytoplasmas were detected. The concentration of bacteria varied from low to high in different sieve tubes (Figs. 1c, d). Fig. 1 SEM images of phytoplasmas (ph) and bacteria (bac) in phloem tissue of field growing (a, d) and indexed (b, c) tomato. Ststarch granes; bac- rod-shaped bacteria; cw-cell wall; cp-cieve pors; sc-salt cristalls of calcium oxalate. Fig. 2 SEM images of virus (vir) conglomerations (a), and rod shaped bacteria (bac) in phloem parenquima of tomato with phytoplasma infection (b). In some specimens prepared from tomato and pepper plants with very strong symptoms of foliar and floral malformations: twisted, wrinkled and spoon like leaves, extremely reduced apical and internodal leaflets, deformed calyx and corolla and virscence of petals, along with phytoplasmas, some rod-shaped bacteria and geminate virus particles were detected in phloem parenchyma (Fig.2, a-b). The presence of viruses in such samples was proved in subsequent assays using molecular techniques: two different begomoviruses were detected and identified [8]. 79

3 Fig. 3 SEM images of infected phloem tissue of pepper. a) phytoplasmas (ph) in a phloem cell; cw-cell wall; b) phytoplasmas (ph) and salt crystalls of calcium oxalate (black arrow) packed in idioblast cell (Id) Fig. 4 Low magnification (x500) SEM image showing a distribution of phytoplasmas (ph) and starch granules (st) in multiple phloem cells. ep-epithelial tissue. High content of starch granules and salt crystals were noted in some phloem cells of diseased tomato and pepper samples (Fig.1, 3-4). The phenomenon of starch accumulation in phloem was reported in many other plant species in the case of phytoplasma and liberibacter infection [3, 6, 10, 11]. 3.2 SEM analysis of citrus species. SEM technique was applied to analyze the possible presence of phytoplasmas and other pathogens in numerous Rutaceae species growing in BCS with multiple yellow type symptoms: chlorosis, leaf dropping, malformations and blotching, necrotic leafstalks and shoots. The similar symptoms were reported in the case of citrus greening (huanglongbing) disease worldwide [11-13]. In phloem tissue of many samples from diseased plants both phytoplasmas and large, up to 2500 nm, baciliform rickettsia-like bacteria (RLB) were detected (Figs. 5-6). The morphology and sizes of bacterial cells were very similar to Candidatus liberibacter detected by SEM in Brazil using more complex technique [14]. In some samples phytoplasma and RLB were observed in the same samples, suggesting the mixed infection with both pathogens (Fig.6b). 80

4 Fig. 5 SEM image of phytoplasmas (ph) in various citrus species: orange (a), mandarin (b) and limonaria (c). sp-sieve pores; ststarch granules; cw-cell wall. Fig. 6 Rickettsia-like bacteria (3,5x 0,5) in phloem of leaf midrib of orange (a); bacteria (bac) and phytoplasmas (ph) in two adjacent phloem cells (b). Fig. 7 SEM images of inclusions of prismatic (p) and needle-like (nlc) crystals in phloem of infected citrus species; (a) orange, (b) and (c) grapefruit. Crystalls of distinct morphological types and compositions were detected in the phloem of citrus plants: prismatic (Figs.7 a,b) and needle-like (Fig.7 b,c). Prismatic crystalls were observed earlier in SEM analysis of plants with yellow type symptoms from different families [6]. High concentration of needle-like and rafidious crystalls was noted in the phloem of species Arecaceae [10]. The functional role of crystalls in infection process is not fully elucidated but there are some speculations about their possible protective role in insects and patogens attacks [15]. Starch granules were also detected in phloem of citrus plants with HLB-like symptoms (Fig.8). High level of starch granules is a phenomenon reported in the case of accumulation of phytoplasmas and liberibacter in phloem tissue [6,10,11]. Though starch is a common component of plant cells, such increasing in concentration is presumedly related with plant defense response mechanisms [10,11,14]. 81

5 Fig. 8 SEM images of accumulation of starch granules (st). (a) limonaria, multiple phloem cells (x750); (b) orange, single phloem cell (x 2500). 4. Conclusions Scanning electron microscopy (SEM) was used successfully to detect phytoplasmas and other bacterial phloem inhabiting pathogens associated with some emerging diseases of agricultural crops. SEM technique enabled for the first time to visualize conglomerations of twisted virus particles in the same samples along with phytoplasmas, thus, to suppose the mixed infection of two distinct pathogens in Solanaceae. In the case of citrus HLB-like disease, phytoplasmas and rickettsia-like bacteria were revealed in phloem tissue, and the mixed infection was diagnosed preliminarily. High concentration of starch granules and crystals in phloem tissue suggest their participation in plant defence response mechanisms during plant-pathogen interaction. Our results demonstrated that the SEM technique is a highly valuable in detection of phloem inhabiting pathogens and is a very important tool in correct diagnosing of emerging yellow-type diseases prior to prove the results by molecular techniques. References [1] Seemuller E, Marcone C, Lauer U, Ragozzino A, Goschl M. Current status of molecular classification of the phytoplasma. Journal of Plant Pathology.1998; 80:3-26. [2] Aljanabi SM, Parmessur Y, Moutia Y, Saumtally S, Dookun A. Further evidence of the association of a phytoplasma and a virus with yellow leaf syndrome in sugarcane. Plant Pathology. 2001; [3] Lebsky V, Poghosyan A. Phytoplasma associated diseases in tomato and pepper in the state of BCS, Mexico: a breaf overview. Bulletin of Insectology, 2007; 60(2): [4] Mauricio-Castillo JA, Arguello-Astorga GR, Ambriz-Granados S, Alpuche-Solis AG. First report of Tomato golden mottle virus on Licopersicon esculentum and Solanum rostratum in Mexico. Plant Disease. 2007; 99:1513 [5] Acosta K, Zamora L, Piñol B, Fernandez A, Chavez A, Flores G, Mendez J, Santos ME, Leyva NE, Arocha Y. Identification and molecular characterization of phytoplasmas asn rickettsia pathogens associated with Bunchy top symptoms (BTS) and Papaya Bunchy Top (PBT) of papaya in Cuba. Crop Protection, 2013; 45(1): [6] Poghosyan A, Lebsky V. Fitoplasmas en cultivos agricolas y plantas silvestres del estado de Baja California Sur. In: Servantes Diaz L, Gonzalez Mendoza D, Grimaldo Juarez O, editors. Los recursos geneticos microbianos y su potencial biotecnologico en la producción agropecuaria en el Noroeste de Mexico. Mexicali, UABC, p [7] Paltrinieri M, Piergiacomi S, Ardizzi S, Contaldo N, Biondi E, Lucchese C, Bertaccini A. Phytoplasma detection and identification in kiwi plants and possible correlation with Pseudomonas syringae pv actinidiae presence. Petria. 2012; 22: [8] Lebsky V, Hernandez-Gonzalez J, Arguello-Astorga GR, Cardenas-Conejo Y, Poghosyan A. Detection of phytoplasmas in mixed infection with begomoviruses: a case study of tomato and pepper in Mexico. Bulletin of Insectology, 2011; 64 (Supplement): [9] de Souza A, da Silva FN, Bedendo IP.A phytoplasma belonging to 16SrIII-A subgroup and dsrna virus associated with cassava frogskin disease in Brazil. Plant Disease. 2014; 98(6): [10] Lebsky V, Oropeza C, Narvaez-Cab M, Poghosyan A. Aplicaciones del MEB para la deteccion de fitoplasmas e inclusiones de almidon y cristales en palma de cocotero (Cocos nucifera) y pritchardia (Pritchardia pacifica). In: Materiales de XI Congreso Nacional de Microscopia; 2012 sep.23-27; San Luis Potosi, Mexico (CD). [11] Achor DS, Etxeberria E., Wang N, Folimonova SY, Chung KR, Albrigo IG. Sequence of anatomical observations in citrus affected with huanglongbing disease. Plant Pathology Journal. 2010; 9(2): [12] Davis MJ, Mondal SN, Chen H, Rogers ME, Brlansky RH. Co-cultivation of Candidatus Liberibacter asiaticus with Actinobacteria from citrus with Huanglongbing. Plant Disease, 1998; 92:

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