Morphology and behavior of gametes and zoospores from the plant-parasitic green , (, )

By Narasinee Thithuan, Penpadsorn Bunjonsiri and Anurag Sunpapao*

Abstract Plant-parasitic in the species Cephaleuros infect leaves, twigs and fruits of numerous host plants worldwide. The reproductive structures of Cephaleuros are important in the infection process. The goal of this study was to determine the in vitro morphology and behavior of the zoospores and gametes of five Cephaleuros species: Cephaleuros karstenii, C. pilosa, C. solutus, C. virescens and Cephaleuros sp. Microscopic observations revealed that zoospores were ellipsoidal, rod-shaped, or spherical with four flagella. Gametes were spherical in shape with two flagella. Zoospores were released from all five Cephaleuros species, but gametes were released only by C. karstenii, C. solutus and Cephaleuros sp. After their release from gametangia, gametes swarmed in a water drop in irregular and circular motions until the resting stage; some gametes attached to each other, and others burst. Zoospores were released from papilla-pores located at the base of zoosporangia and swarmed in a water drop in irregular and circular motions. Some zoospores germinated and others burst, similar to the gametes. In this study, germinated zoospores formed germ tubes, and filaments containing carotenoid pigment, and then died without forming thalli.

*Corresponding Author E-mail: [email protected]

Pacific Science, vol. 73, no. 3 Mayr 17, 2019 (Early view) Introduction

Green algae in the genus Cephaleuros Kunze ex E.M. Fries are plant parasites belonging to the order , family (Guiry and Guiry 2017). They are subaerial algae that create circular lesions on leaves, twigs and fruits of numerous herbaceous and woody plant species worldwide (Printz 1939, Joubert and Rijkenbberg 1971, Thompson and Wujek 1997,

Pitaloka et al. 2015, Sunpapao et al. 2015). Moderate to severe damage is found on tea (Camelia sinensis (L.) Kuntze) plantation crops (Marlatt and Alfieri 1981, Thompson and Wujek 1997,

Suto and Ohtani 2009) and in guava (Psidium guajava Linn.) orchards in northern and southern

Thailand (Sunpapao et al. 2016a). The growth of Cephaleuros on leaves causes a loss of photosynthetic area due to necrosis of tissues beneath or around the algal thalli (Safeeulla and

Govindu 1948). In Thailand, eleven species of Cephaleuros have been described recently:

Cephaleuros diffusus, C. druetii, C. expansa, C. henningsii, C. karstenii, C. lagerheimii, C. parasiticus, C. pilosa, C. solutus, C. tumidae-setae and C. virescens (Sunpapao and Pitaloka

2015, Pitaloka et al. 2015, Sunpapao et al. 2015, 2016a, 2016b, 2016c, 2017; Wonglom et al.

2018). However, details of their parasitism are still unknown.

Reproduction of Cephaleuros species can be either asexual or sexual. Asexual reproduction produces zoospores with four flagella (quadriflagellate) in zoosporangia. Sexual reproduction produces gametes with two flagella (biflagellate) within gametangia. The gametangia are globular to irregular cells that develop from the ends of the main filaments of an algal thallus (Thompson and Wujek 1997). The gametes and zoospores are considered the active entities that infect host plants (Mann and Hutchinson 1907, Wolf 1930, Suématu 1962,

Chowdary and Jose 1979). Both types of reproductive cells are able to swim in free water on the surface of the host plant, penetrate the host cuticle and generate disc-like algal thalli (Joubert and

Rijkenberg 1971). The algae deplete the surrounding water and mineral nutrients of the host

2 (Wolf 1930). The reproductive cells of Cephaleuros species need water in order to spread within the host. However, the morphology and behavior of reproductive cells after their release from reproductive organs still remain unknown. The goal of this research was to increase our knowledge of the morphology and behavior of the gametes and zoospores of five Cephaleuros species in Thailand.

MATERIALS AND METHODS

This study was conducted in the Pest Management Department field, Faculty of Natural

Resources, Prince of Songkla University, Hatyai, Thailand. Algal thalli with mature reproductive structures were collected from leaves of muricata, Garcinia mangostana, Mangifera indica, Murraya paniculata, Piper nigrum and Sandoricum koetjjape. Cephaeluros morphospecies were identified using the key to species by Thompson and Wujek (1997).

Microscopic features of asexual and sexual reproductive structures were observed under both stereo (Leica S8AP0, Leica, Germany) and light (Leica DM750, Leica, Germany) microscopes.

The release of gametes and zoospores from gametangia and zoosporangia was examined twice, in 2015 and again in 2016. Algal thalli 2–3 mm in diameter were removed from plants with a razor blade and placed in a drop of sterile water on a glass slide, without a cover slip.

Reproductive cells (n=30) from 5 thalli (total 150 cells) of each Cephaleuros species were observed under the light microscope. Conjugation of gametes was examined immediately after release from gametangia. Another set of reproductive cells was then observed for the germination. A glass slide with a water drop containing both gametes and zoospores was incubated in a covered Petri dish with moist paper to maintain humidity at ambient temperature

(28±2C) with a 12-hour light and dark photoperiod. Germination of gametes and zoospores in the water drop was observed for 2–7 days with the light microscope.

3 RESULTS

Observation of reproductive structures

We found Cephaleuros species on the leaves of all five plant taxa examined: C. karstenii on A. muricata, C. pilosa on G. mangostana, C. solutus on S. koetjape, C. virescens on M. indica, and

Cephaleuros sp. on M. paniculata and P. nigrum. Both asexual and sexual structures were observed (Fig. 1). Zoospores were present in the zoosporangia in numbers that differed by species (Table 1). Zoospores of C. karstenii, C. virescens and Cephaleuros sp. (on M. paniculata) were ellipsoidal and measured 5.37±0.21 × 6.76±0.34, 5.62±0.49 × 6.89±0.47, and

5.18±1.13 × 6.04±1.04 µm, respectively. The zoospores of C. pilosa and Cephaleuros sp. (on P. nigrum) were rod-shaped and measured 2.91±0.85 × 9.63±0.78 and 2.88±0.56 × 9.72±0.18 µm, respectively. Zoospores of C. solutus were spherical and measured 9.68±0.52 × 9.59±0.64 µm.

Sphere-shaped gametes were only observed from the gametangia of C. karstenii, C. solutus and

Cephaleuros sp. (on P. nigrum) and measured 5.08±0.93 × 5.75±1.07, 4.58±0.52 × 5.03±1.94 and 5.76±1.46 × 5.58±0.81 µm, respectively (Table 2).

Behavior of gametes and zoospores in a water drop

Gametes and zoospores were released 5–10 min after thallus fragments were placed in a cool

(10C) water drop. After release, zoospores of the five Cephaleuros species swarmed actively for

1–5 min, performing irregular circular movements. Many zoospores of all five species then lost their flagella and burst, but some remained active. The active zoospores of C. solutus and C. virescens germinated and formed germ tubes in a water drop 24 h after release from their zoosporangia. After their release from gametangia, gametes of the five Cephaleuros species swarmed in a water drop for 10–30 min, then some burst whereas others remained active.

4 Germination of gametes and zoospores

In our experiments, gametes released from gametangia of C. karstenii and the Cephaleuros sp. from P. nigrum attached to each other but did not appear to conjugate in a water drop (Fig. 4C).

Zoospores of C. solutus and C. virescens germinated following release from their zoosporangia

(Fig.5A–G), and then stopped after swarming. Zoospores of C. solutus were swollen, spherical to irregular in shape, 5–9 m in diameter, with germ tubes 2.91 ± 0.56 m long (Fig. 5B) beginning to develop at their apex (Fig. 5A and B). Then the germ tubes formed unbranched filaments

11.43 ± 0.68 m long after 4 days. Yellow-orange carotenoid pigments were produced in the young filaments of C. solutus 5 days after zoospore release (Fig. 5E–F). For C. virescens, the germ tube was 2.31 ± 0.43 m long 7 days after release from zoosporangia, and it was the same length as those of C. solutus after 4 days (Fig. 5C–D), and then died (Fig. 5G). The morphological characteristics of the germ tubes did not differ among the Cephaleuros species in this study and no germination of gametes was observed.

DISCUSSION

In this study, released gametes were spherical in shape and zoospores were ellipsoidal, rod- shaped, or spherical. Previous reports described gametes and zoospores as ellipsoidal to fusiform, but spherical when they lost their flagella (Suto and Ohtani 2009). However, we found only spherical gametes, and zoospore shapes varied among species (Fig. 2). This characteristic has been rarely reported in studies on the Trentepohliales and might be useful for species identification. When released, the zoospores appeared to have two flagella because the four flagella were twisted into two pairs.

In this study we investigated the behavior of gametes and zoospores in a water drop. The results showed gametes attached to each other but there was no evidence of sexual conjugation

5 of gametes (Fig. 4C) for C. solutus and Cephaleuros sp. from P. nigrum. No conjugation of gametes has been observed for C. virescens (Chowdary and Jose 1979, Suto and Ohtani 2013).

Gametes and zoospores were not released until their gametangia and zoosporangia were placed in water. Release of gametes and zoospores required not only water but also mechanical shock (Suto and Ohtani 2013). This suggests that water is needed to release reproductive cells of

Cephaleuros species. We also noticed that several gametes and zoospores burst immediately after release, similar to a previous report by Suto and Ohtani (2013). The reason that the cells burst remains unknown.

Following germination, the young filaments of C. solutus produced orange carotenoid pigments sometimes referred to as hematochrome (Wolf, 1930). The germination of Cephaleuros in a water drop has been reported in five Cephaleuros species from Japan: C. aucubae, C. biolophus, C. japonicus, C. microcellularis and C. virescens (Suto and Ohtani 2013). The authors demonstrated different percentages of germination for the five Cephaleuros species

(Suto and Ohtani 2013). However, there are no previous reports on germination of zoospores from C. solutus. In our study, the germ tubes of C. solutus formed unbranched filaments, whereas germ tubes of C. virescens germinated and died (Fig. 5). However, the young filaments of C. solutus then collapsed and withered before forming thalli. This finding suggests that although the reproductive cells were able to germinate or attach in free water (e.g., a water drop) outside the host plant, they may need to access the host tissues to obtain the nutrients necessary for survival and growth.

In this study, we described the release of zoospores and gametes, their motion in a water drop and initial germination of five Cephaleuros species. However, host penetration, establishment and formation of reproductive structures, which are important steps in the

6 infection process are still remained unknown. To understand these phenomena in nature, further analysis on host establishment of Cephaleuros species is needed.

ACKNOWLEDGMENTS

The authors thank Prince of Songkla University (grant no. NAT590712S and NAT610511N) and the Center of Excellence in Agricultural and Natural Resources Biotechnology (CoE-ANRB) phase 2, Faculty of Natural Resources, Prince of Songkla University, Thailand for funding and support for this project. The copy-editing service of RDO/PSU and the helpful comments of Dr.

Seppo Karrila are gratefully acknowledged.

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9 Table 1 General Characteristics of Cephaleuros Zoospores Cephaleuros Host plant Number of Shape Width1 Length1 species zoospores (m) (m) C. karstenii Annona muricata 24–49 ellipsoidal 5.37±0.21 6.76±0.34 C. pilosa Garcinia 3–8 rod 2.91±0.85 9.63±0.78 mangostana C. solutus Sandoricum 21–44 spherical 9.68±0.52 9.59±0.64 koetjape C. virescens Mangifera indica 21–47 ellipsoidal 5.62±0.49 6.89±0.47 Cephaleuros sp. Murraya paniculata 18–31 rod 2.88±0.56 9.72±0.18 Piper nigrum 4–7 ellipsoidal 5.18±1.13 6.04±1.04 1 Average of 30 (n = 30)

Table 2 General Characters of Cephaleuros Gametes Cephaleuros Host plants Number of Shape Width1 Length1 species gametes (m) (m) C. karstenii Annona muricata 27–51 spherical 5.08±0.93 5.75±1.07 C. pilosa Garcinia –2 – – – mangostana C. solutus Sandoricum koetjape 11–34 spherical 4.58±0.52 5.03±1.94 C. virescens Mangifera indica – – – – Cephaleuros sp. Murraya paniculata – – – – Piper nigrum 114–155 spherical 5.76±1.46 5.58±0.81 1 Average of 30 spores (n = 30) 2 not detected

10 Figure 1. Characterization of the reproductive structures of Cephaleuros spp. A–F) asexual reproductive structure: A) lesion on host is composed of prostrate and erect systems; B) detail of asexual reproductive structure composed of sporangiophores (sp), suffultory cells, head cells (hc) and zoosporangia (zs); C) papilla-pore at the base of a (arrow); D) zoospores in zoosporangia; E) zoosporangia releasing zoospores; F) quadriflagellate zoospores in the resting phase. G–K) sexual reproductive structure: G) gametangia, H) release of gametes from a gametangium, I) biflagellate gametes, J) attached gametes, and K) burst gametes. 11 Figure 2. Morphology of asexual reproductive cells (zoospores) and sexual reproductive cells

(gametes) of Cephaleuros species: A) ellipsoidal, rod- and spherical-shaped, quadriflagella zoospores; and B) spherical, biflagellate gamete. Scale bar = 10 m.

12 Figure 3. Germination and development of Cephaleuros zoospores in water: A) zoospores released from papilla-pore at the base of sporangia; B) zoospores swim with irregular and circular motions; C) zoospores stop movement, release their flagella D) zoospores that do not burst, form a germ tube at their apex, and E) the germ tube elongates and produces orange pigment. Scale bar = 10 m.

13 Figure 4. Behavior of Cephaleuros gametes in a water: A) gametes released from papilla-pore of gametangia, B) gametes swim in a water drop, C) some gametes burst while others stuck together (arrow).

14 Figure 5. Germination of Cephaleuros zoospores in water: A) zoospore one day after release, B) zoospore germination, C) zoospore forms a filament (arrow), D) deformation of zoospore, E) first cell of a young filament, F) a young filament seven days after germination of the sporangia, and G) desiccated filamentous cell. Scale bar = 10 m.

15