Scanning electron microscopy detection of seed-borne fungi

Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)

Scanning electron microscopy detection of seed-borne fungi in blotter test Marcelo de Carvalho Alves *1 and Edson Ampélio Pozza2 1 2

Federal University of Mato Grosso, Soil and Rural Engineering Department, Brazil Federal University of Lavras, Plant Pathology Department, Brazil

Scanning electron microscopy (SEM) was used to detect seed-borne fungi in seeds submitted to blotter test. Two different conditions of blotter test and water restriction treatment were evaluated. In the blotter test, seeds were subject to conditions that enabled pathogen growth and expression, while the water restriction method consisted in prevent seed germination during the incubation period, resulting in the artificial inoculation of fungi. In the first condition, seeds of common bean (Phaseolus vulgaris L.), maize (Zea mays L.) and cotton (Gossypium hirsutum L.) were submitted to the standard blotter test and then prepared and observed with SEM. In the second condition, seeds of cotton (G. hirsutum), soybean (Glycine max L.) and common bean (P. vulgaris L.) were respectively inoculated with Colletotrichum gossypii var. cephalosporioides, Colletotrichum truncatum and Colletotrichum lindemuthianum by the water restriction technique, followed by preparation and observation with SEM. The standard SEM methodology was adopted to prepare the specimens. Considering the seeds submitted to the blotter test, it was possible to identify Fusarium sp. on maize, C. gossypii var. cephalosporioides and Fusarium oxysporum on cotton, Aspergillus flavus, Penicillium sp., Rhizopus sp. and Mucor sp. on common bean. Structures of C. gossypii var. cephalosporioides, C. truncatum and C. lindemuthianum were observed in the surface of inoculated seeds. Keywords Fungi; Seed; Pathology; Identification; SEM; Health Analysis

1. Introduction Seed occupies a small niche in the overall agricultural economy, but seeds with good quality are the basis of all agricultural production. Seed-borne fungi affect the quality of seeds at all stages of production, from the cropping stages until post-harvest, processing, storage and marketing [1]. The economical importance of seed-borne fungi in the agroecosystems could be explained by the survival and spread of pathogens in the seeds for long periods of time, reducing its germination, vigour, accelerating the deterioration in storage, introducing pathogens into new areas and increasing the inoculum source in the field. If the primary inoculum transported by seeds, finds environmental suitability and host susceptibility, initial outbreaks can be established, and from there, secondary inoculum sources can be produced in lesions of infected plants, causing variability of disease along the space and time [2, 3]. Fungi are the largest group of the seed-borne pathogens investigated by seed pathologists due to its capacity of multiplication and survival in nature [4, 5, 6]. Considering that some seed health methods are not successful in the detection of some fungal species, the fungi could be grouped according to their parasitic nature and biological evolution, in relation to seed health testing methods (Table 1) [7]. Seed health testing is routinely carried out in most countries for seed certification, quality assessment and plant quarantine. To ascertain the existence of seed-borne fungi there are several methods of detection that differ in sensitivity, equipment and materials [8]. Among these methods, the blotter test is one of the most used, applied to all types of seeds, including seeds for cereals, vegetables, ornamentals and forests. The blotter test is effective to detect a large number of seed-borne fungi in lots of seeds, supported by the utilization of light microscopy and stereomicroscopy as equipment for visualization of structures and signals of fungi [9]. Moreover, techniques of observation of microorganisms with light microscope and stereomicroscopy can be supplemented by alternative methods with greater precision, such as electron microscopy, often employed to study plants, algae, fungi, soil micro-organisms, seeds, fruits, pollens, spores [10], among other applications. This may be justified, because the introduction of scanning electron microscopy (SEM) has caused a revolution in the study of the microscopic world, considering advantages such as high depth of field giving three-dimensional aspect to the images, large magnitude of increase from 10 to 1,000,000 times, rapid processes of image digitalization and acquisition, easiness to prepare and operate samples, as well as accessible costs [11]. The images generated by SEM were also used to enlarge the possibilities of teaching-learning interaction based on the application of virtual reality techniques for microorganisms’ visualization [12] and to study details of microorganisms’ taxonomy [13]. According to Machado et al.[6], detection of various formae specialis of fungi is a challenge in Seed Pathology. In this context, the objective of the present study was to evaluate the application of standard SEM methodology as an alternative to identify seed-borne fungi in seeds submitted to the blotter test.

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Table 1 Fungi groups according to the parasitic nature and biological evolution in relation to recommended seed health testing methods (Adapted from Machado et al.[7]). Biotrophic group

Necrotrophic group Higher taxon

Lower taxon

Higher taxon

Bremia Peronospora Plasmopara Sclerospora

Ustilago Tilletia Puccinia

WASHING AND EMBRYO METHODS

Lower taxon Rhizopus

-

Ascomycotina Sclerotinia Diaporthe Glomerella Pleospora Gibberella Pyrenophora

Mitosporic fungi Phoma Phomopsis Diplodia Septoria Ascochyta Botryodiplodia Macrophomina Colletotrichum Rhizoctonia Sclerotium

Fusarium Phaeoisariopsis Aspergillus Penicillium Cercospora Botrytis Bipolaris Drechslera Pyricularia Verticillium

INCUBATION STANDARD METHODS (agar and blotter test) SPECIFIC METHODS (e.g. roll paper, fluorescent techniques, selective substrates etc.)

2. Materials 1. Materials for blotter test incubation method for seed health analysis 8 repetitions of 50 seeds for each species; absorbent paper; petri dishes or equivalent containers; transparent lid for light; NUV light; freezer; sodium hypochorite solution at 1% concentration; 2,4-D salt (sodium 2,4-dichlorophenoxyacetate) at 5-10 ppm concentration; incubator with temperature and light regulation; agar 0.2%; laminar flow hood; autoclave; dishwasher; microscopes, compound and stereoscopic. 2. Materials for blotter test incubation method with water restriction for seed health analysis - The same as protocol for blotter test, but mannitol or other osmotic compound at osmotic potentials of -0,6 to -1,0 MPa is incorporated in the blotter substrate to prevent seed germination, instead of freezer or 2,4-D salt. 3. Materials for scanning electron microscopy observation Blade; eppendorff tubes; modified Karnovisk’s solution (Glutaraldehyde 2.5%, formaldehyde 2.5% in cacodilato sodium buffer 0.05 M, pH 7.2, CaCl2 0.001M); buffer cacodilato 0.05 M; tetroxide solution of 1% of osmium in buffer cacodilato 0.05 M pH 7.2; distilled water; gradient of acetone (25, 50, 75, 90 and 100%); CPD Balzers 030® equipment; adhesive tapes; metal stubs; aluminium paper; gold; SCD Balzers 050® equipment; scanning electron microscope LEO Evo 40®; digital image processing software LEO User Interface®.

3. Methods 1. Protocol for blotter test incubation method for seed health analysis [13]. In the first experiment, seeds of common bean (Phaseolus vulgaris L.), maize (Zea mays L.) and cotton (Gossypium hirsutum L.) were submitted to the standard blotter test. The blotter test preparation was conducted in accordance with Machado et al.[8] criteria. The tests were prepared with 8 repetitions of 50 seeds for each species. Seeds of maize were incubated initially 24 hours in room condition, then kept 24 hours in a freezer (-20 ºC), followed by 5 days under NUV light, with 12 hour photoperiod, at 20 ºC. To prevent seed germination of common bean and cotton during the incubation period, the substrate paper was moistened with 2,4-D salt solution 5-10 ppm and 0.2% agar to prevent seeds to roll during handling. After that, seeded dishes were placed under NUV light with 12 hour photoperiod at 20 ºC per 7 days. Seeds were individually examined with a stereomicroscope at 30-80 magnification and slide mounts were prepared for observation using light microscope, in order to confirm fungi identity.

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2. Protocol for blotter test incubation method with water restriction for seed health analysis [13]. In the second experiment, seeds were inoculated by the water restriction technique [14]. Lots of healthy seeds were chosen in order to ensure the occurrence of the inoculated fungal species. The pathogens were obtained from lots of infected seeds in the Laboratory of Seed Pathology of Federal University of Lavras. The pure colony of each pathogen was scraped with a Drigalsky loop in order to obtain a suspension of conidia and mycelium with 1 ml solution. The solution was transferred to petri dishes of 15 cm in diameter, with potato dextrose agar (PDA) + mannitol medium, at 1.0 MPa. Mannitol concentrations were obtained through Van't Hoff equation [15]. The petri dishes were incubated in chamber with 20º C and photoperiod of 12 hours, for five days. The seeds were disinfected in a solution of sodium hypochlorite 1% per 3 minutes, then washed with sterile water and dried in the shade per 24 hours. Then, 35 grams of seeds of cotton (G. hirsutum), soybean (G. max) and common bean (P. vulgaris) were distributed in single layer on the colony of Colletotrichum gossypii var. cephalosporioides, Colletotrichum truncatum and Colletotrichum lindemuthianum, respectively. A suspension of conidia with concentration of 1x106 was sprayed on the single layer of seeds to ensure the inoculation. After that, the plates returned to the chamber of 20 ºC with photoperiod of 12 hours, per 144 hours. 3. Protocol for scanning electron microscopy observation [13]. Seeds of the first and second experiment were prepared to be observed in SEM using a standard procedure. Seeds were segmented with blade in thin layers of no more than 0.5 to 1 cm. The segments were immersed in eppendorff tubes containing 1.5 ml of modified Karnovisk’s solution (Glutaraldehyde 2.5%, 2.5% formaldehyde in cacodilato sodium buffer 0.05 M, pH 7.2, CaCl2 0.001M) by 24 hours. After that, samples were washed in aldehyde by 3 times of 10 minutes in buffer cacodilato 0.05 M, followed by immersion in tetroxide solution of 1% of osmium in buffer cacodilato 0.05 M pH 7.2, at room temperature, in laminar flow chamber, per 4 hours. After this period, the samples were washed 3 times in distilled water and then dehydrated in gradient of acetone (25, 50, 75, 90 and 100%), remaining 10 minutes in each concentration, being repeated by 3 times in the solution of 100%. Subsequently, the samples were submitted to CPD Balzers 030® equipment to complete dehydration. The specimens were pasted using adhesive tapes on the surface of stubs covered with aluminium and submitted to the metallization with gold using SCD Balzers 050® equipment, in order to increase its conductivity. Finally the specimens were observed by the scanning electron microscope LEO Evo 40®, interfaced by digital image processing software [16]. The digital images were generated, filtered and recorded in a computer. After image acquisition, it was proceeded image enhancement, combining brightness, contrast and intensity [16].

4. Results and Discussion The studied organisms were classified in the fungi kingdom [17], amastigomycota division, zygomycetes class for Mucor sp. and Rhizopus sp. and deuteromycetes class, for Colletotrichum, Fusarium, Aspergillus and Penicillium [18]. From the first test, conducted with seeds of cotton, common bean and maize submitted to the blotter test, the following results were obtained: In surface of cotton seeds, after laboratory diagnosis of Fusarium oxysporum and the use of SEM methodology, there were presences of mycelium and oval reniformes microconidia with 5-12 x 2.2-3.5 μm. False head of microconidia were verified in small conidiophores along the hifae (Figure 1), similarly as related by Machado et al. [6], visualizing this fungus through estereomicroscope and microscope. Gomes et al. [19] also used the SEM technique to study the effect of vicilina of Vigna unguiculata in germination and development of espores of F. oxysporum. According to the authors, the SEM enabled to observe microconidia and mycelium, similar to this study. Still referring to the surface of cotton seeds, but after the laboratory diagnosis of Colletotrichum gossypii var. cephalosporioides and the use of SEM methodology, there were setae arising directly from the seed coat, in a stromatic structure and aerial mycelium covering the seed. Small conidial clusters could be seen in the apex of setae remain adhered look like “heads”. Conidia occurred in small groups, cylindrical, tapered towards one end, measuring 3.5-7x1225 μm, as reported by Tanaka et al. [20] and Machado et al. [6] (Figure 2). Regarding common bean seeds, it could be observed on seed surface, after laboratory diagnosis, Penicillium sp., Aspergillus flavus, Rhizopus sp. and Mucor sp. Observing Penicillium sp. structure by the SEM procedure, conidiophores were 30-100 x 4-5 μm, phialides with 15-28 x 3-5 μm, conidia usually elliptical, 3,4-12 x 3-8 μm (Figure 3). In the case of Aspergillus flavus, it was observed colony with 500-600 μm, conidia globose to subglobose, 3-6 μm in diameter, similarly as related by Machado et al. [6] (Figure 4). In the case of Rhizopus sp. there was observed sporangiophores, rhizoids and sporangium, with marked differences of multispored sporangium of Mucor sp. (Figure 5). The seeds of maize, after laboratory diagnosis of Fusarium verticillioides, presented colony with phialides and microconidiation with conidium at the phialide apex, as reported by Machado et al. [6] as characteristics of the pathogen (Figure 6). Glenn et al. [21] also observed F. verticillioides conidiation using SEM methodology. Guarro et al. [22] studying F. verticillioides isolated from blood at magnification of x2,320 also observed microconidia clavate with a slightly truncate base, produced from monophialides. Hirata et al. [23] studying the molecular characterization of F.

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verticillioides from rotten banana imported into Japan, observed clavate aerial conidia formed from apical tips of monophialides in false heads and branched aerial conidiophores bearing strictly monophialides, from which clavate aerial conidia were formed in chains, as well as sparcely branched sporodochial conidiophores and conidia, similar to the observed in the present work.

Fig. 1 Scanning electron microscopy of cotton seed surface with laboratory diagnosis of Fusarium oxysporum, presenting microconidia (upper left and right) false heads of microconidia formed on short conidiophores along the hyphae (middle left), terminal and intercalary phialides (middle right), clusters of conidia (down left) and hyphae (down right).

Fig. 2 Scanning electron microscopy of cotton seed surface with laboratory diagnosis of C. gossypii var. cephalosporioides, presenting acervuli (upper left), aerial mycelium with fertile setae (upper right), injury on seed surface covered with acervuli and mycelium (middle left), conidia produced on short conidiophores on the acervuli (middle right) and also on the top of some setae (down left), setae produced out on the acervuli, single, erect, exhibiting ramifications at the top (down right).

In the second experiment, in which seeds of cotton (G. hirsutum), soybean (G. max) and common bean (P. vulgaris) were inoculated with Colletotrichum gossypii var. cephalosporioides, Colletotrichum truncatum and Colletotrichum lindemuthianum, respectively, it was possible to visualize conidia and mycelium of C. Gossypii var. cephalosporioides on cotton surface (Figure 7), acervuli of C. truncatum on soybean surface (Figure 8) and setae of C. lindemuthianum on

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common bean surface (Figure 9). In all inoculated situations, it was possible to observe fungi lesions in the seeds, confirming the potential to use the water restriction technique for inoculation of Colletotrichum in seeds of cotton, soybean and common bean. Jackowiak et al. [24] also verify the possibility of inoculation of wheat (Triticum aestivum) seeds with conidia of Fusarium culmorum, using SEM methodology. Lins et al. [25] also used SEM to follow the process of infection, colonization and reproduction of different isolates of Colletotrichum spp. in coffee plantlets, obtained by embryo culture.

Fig. 3 Scanning electron microscopy of common bean seed surface with laboratory diagnosis of Penicillium sp., presenting colony aspect (upper and middle left), conidiophores and phialides (upper, middle and down right), conidia subglobose to cylindrical (down left).

Fig. 4 Scanning electron microscopy of common bean seed surface with laboratory diagnosis of Aspergillus flavus, presenting colonies with conidiophores and conidial heads (upper left and right, middle left), conidial heads typically radiate, splitting into several poorly defined columns, occasionally columnar heads (middle right), conidia are globose to subglobose, conspicuously echinulate, sometimes elliptical or pyriform (down left and right).

Based on results of comparative seed health analysis realized by ISTA members, the human factor is the cause of greatest discrepancy between the results of tests. In general, lack of practice of analysts is the main cause of variation of these tests [26]. Machado et al. [7] also related that for groups of fungi represented by biotrophic and necrotrophic

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species, specific or more selective methods are required for their reliable detection in routine analysis of various formae specialis of fungi such as Fusarium, Colletotrichum, Diaporthe (Phomopsis). Thus, considering the advantages of SEM-related characteristics such as high depth of field, large magnitude of increase, fast image digitalization and acquisition, easiness of preparation and operation of samples, as well as relatively accessible costs, this approach may become a viable contribution to decision support in routine seed health analysis. Despite of that, Blakemore and Reeves [27], studying the perspectives of the use of modern techniques in seed health testing, did not included the use of scanning electron microscopy as alternative to minimize the problems of seed health tests to ensure correct diagnoses.

Fig. 5 Scanning electron microscopy of common bean seed surface with laboratory diagnosis of Mucor sp. presenting sporangiophore and multispored sporangium in the center of the image (upper left), sporangiospores (upper right, down left) and Rhizopus sp. with stolon, rhizoids, sporangiophores and sporangium (down right).

According to the authors, seed health testing requires simple and quickly tests for certification and the use of nucleic acid techniques are the unique example of modern techniques that can help with the problems of traditional seed health tests.

Fig. 6 Scanning electron microscopy of cotton seed surface with laboratory diagnosis of Fusarium oxysporum, presenting microconidia (upper left and right) false heads of microconidia formed on short conidiophores along the hyphae (middle left), terminal and intercalary phialides (middle right), clusters of conidia (down left) and hyphae (down right).

Although nucleic acid techniques have proved to present several advantages when compared to traditional techniques, the results of genoma analysis do not give an indication about viability of inoculums present in seeds and the results are qualitative and not quantitative. According to Carvalho Vieira and Machado [28], a good strategy to implement seed health tests based on genoma analysis, is the application of these methods to make an initial screening during seed examination by conventional methods.

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Fig. 7 Scanning electron microscopy of cotton seed surface inoculated with C. gossypii var. cephalosporioides, presenting mycelium (upper left and right), acervuli and longer setae emerging from the seed coat and produced in small numbers (middle left), conidia cylindrical, tapering towards one end (middle right and down left and right).

Fig. 8 Scanning electron microscopy of soybean inoculated with Colletotrichum truncatum, presenting mycelium (top left) and acervuli and mycelium on the seed surface (top right, middle left and right and down left and right).

Therefore, considering the results of the present work, SEM methodology could also be useful in certification programs, mainly in the cases where is necessary to make differentiation of closely related species or other forms of variation during seed examination by conventional methods. SEM methodology has also advantages when compared to genome techniques, considering that small structures of the fungi can be measured and quantified by the images stored in the computer, for further confirmation of the report obtained during the analysis of seeds by the conventional methods. In the present study, SEM methodology enabled to observe the interaction of fungi on surface of seeds of cotton, common bean, soybean and maize, presenting potential to increase the opportunities for teaching and learning in Seed Pathology, depending on the level of detail of observed structures. The adoption of this technique in the future of seed

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health analysis routine could be useful to identify fungal structures that enable ensuring the implementation of correct diagnoses as well as to facilitate conduction of more detailed taxonomic classification of seed-borne fungi.

Fig. 9 Scanning electron microscopy of common bean inoculated with Colletotrichum lindemuthianum, presenting colony (top left), mycelium and acervuli (top left and right and middle right), conidia cylindric with rounded ends (middle right and down left and right).

5. Conclusions Scanning electron microscopy methodology was a potential alternative for study and identification of seed-borne fungi in seeds of common bean (Phaseolus vulgaris L.), maize (Zea mays L.) and cotton (Gossypium hirsutum L.) after blotter test preparation. Scanning electron microscopy enabled to observe fungi lesions and fungi signals in seeds of cotton (Gossypium hirsutum L.), soybean (Glycine max L.) and common bean (Phaseolus vulgaris L.) inoculated by water restriction technique associated with Colletotrichum gossypii var. cephalosporioides, Colletotrichum truncatum and Colletotrichum lindemuthianum, respectively. The adoption SEM methodology in blotter test analysis could be useful to identify fungal structures that enable ensuring the implementation of correct diagnoses as well as to facilitate conduction of more detailed taxonomic classification of seed-borne fungi. Acknowledgements The support by Prof. Eduardo Alves and the Laboratory of Ultra-structural Analysis and Electron Microscopy, Plant Pathology Department, Federal University of Lavras are gratefully acknowledged.

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[12] Sforza PM, Eisenback JD. The Virtual Nematode. ISPP-ITSymposium. New Zeland: Symposium covering Instructional Technology in Plant Pathology; 2001. [13] Alves MC, Pozza EA. Scanning electron microscopy applied to seed-borne fungi examination. Microscopy Research and Technique. 2009;72:482-488. [14] Machado JC, Guimarães RM, Vieira MGGC, Souza RM. Use of water restriction technique in seed pathology. Seed Testing International. 2004; 128:12-16. [15] Salisbury FB, Ross CW. Plant Physiology. Belmont: Wadsworth; 1991. [16] Gonzalez RC, Woods RE. Digital image processing. NJ: Prentice Hall, Englewood Cliffs; 2002. [17] Whittaker RH. New concepts of kingdoms of organisms. Science. 1969;163:150-160. [18] Alexopoulos CJ, Mims CW. Introductory mycology. New York: John Wiley & Sons, 1979. [19] Gomes VM, Cunha MD, Miguens FC, Fernandes KVS, Rose TL, Xavier-Filho J. Ultrastructure and immunolabelling of fungi cells treated with Vigna unguiculata vicilins (7S storage proteins). Plant Science. 1998;138:81-89. [20] Tanaka MAS, Menten JOM, Machado JC. Growth habit of Colletotrichum gossypii and C. gossypii var. cephalosporioides on cotton seeds. Bragantia. 1996;55:95-104. [21] Glenn AE, Richardson EA, Bacon CW. Genetic and morphological characterization of a Fusarium verticillioides conidiation mutant. Mycologia. 2004;96:968-980. [22] Garro J, Nucci M, Akiti T, Gené J. Mixed infection caused by two species of Fusarium in a human immunodeficiency viruspositive patient. Journal of Clinical Microbiology. 2000;38:3460-3462. [23] Hirata T, Kimishima E, Aoki T, Nirenberg HI, O’Donnell K. Morphological and molecular characterization of Fusarium verticillioides from rotten banana imported into Japan. Mycoscience. 2001;42:155-166. [24] Jackowiak H, Packa D, Wiwart M, Perkowski J. Scanning electron microscopy of Fusarium damaged kernels of spring wheat. International Journal of Food Microbiology. 2005;98:113-123. [25] Lins SRO, Abreu MS, Alves E. Histopathologic study of Colletotrichum spp. in coffee plantlets. Fitopatologia brasileira. 2007;32:488-495. [26] Neegaard P. Seed pathology. MacMillan Press, London; 1979. 839 p. [27] Blakemore EJA, Reeves JC. Perspectives of the use of modern techniques in seed health testing. In: Seed-borne fungi: a contribution to routine seed health analysis. Zurich: International Seed Testing Association; 2002. pp. 19-22. [28] Carvalho Vieira MGG, Machado JC. Applicability of molecular techniques for detection of seed-borne fungi under certification. In: Seed-borne fungi: a contribution to routine seed health analysis. Zurich: International Seed Testing Association. 2002; pp. 82-91.

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Scanning electron microscopy detection of seed-borne fungi

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