Salvia Officinalis

Biologia 65/4: 603—608, 2010 Section Botany DOI: 10.2478/s11756-010-0060-4

Characteristics of Phomopsis sclareae obtained from sage (Salvia oﬃcinalis)

Beata Zimowska

Department of Plant Pathology, University of Life Sciences in Lublin, Leszczy´nskiego 7,PL-20-069 Lublin, Poland; e-mail: [email protected]

Abstract: In the years 2004–2006 the species P. sclareae was isolated from sage stems showing the symptoms in the form of bark peeling oﬀ and breaking. On the basis of 5 isolates randomly chosen from the fungus population, morphology and the conditions of temperatures favourable for the most intensive growth and creation of the fungus infectious material were studied. The temperature of −6 ◦C was viewed as unfavourable for the fungus growth, and that of 32 ◦C was considered to prevent the formation of picnidia and conidia. The dynamic growth of the colonies and the formation of numerous picnidia and conidia were observed at the temperatures ranging from 20 ◦Cto28◦C. Key words: Salvia oﬃcinalis; Phomopsis sclareae; morphology; sage

Introduction & Machowicz-Stefaniak 2004). The obtained fungi colonies, after segregation and creation of one-spore cultures, were Intensive research on herbs’ healthiness conducted divided into species on the maltose medium and on stan- in recent years in south-eastern Poland has pro- dard media (Ramirez 1982; Nelson et al. 1983; Uecker 1988; vided information on fungi species, not registered Gruyter J De; Noordeloos 1992). The obtained isolates of P. sclareae were identified on in Poland, which colonise and damage various plant PDA medium (Uecker 1988; Coelho et al. 1997). For further organs (Zimowska 2004; Zimowska & Machowicz- research, five isolates of P. sclareae were randomly chosen Stefaniak 2005; Zimowska 2007; Zimowska 2008b; Za- from the fungus population: S 584, S 632, S 792, S 1102 lewska 2008; Machowicz-Stefaniak et al. 2008). Among and S 1107. Each isolate grew in the following tempera- these was Phomopsis sclareae Sawar. (Uecker 1988), tures: −6 ◦C, 5 ◦C, 10 ◦C, 16 ◦C, 20 ◦C, 24 ◦C, 28 ◦Cand32◦C. which was isolated from the stems of sage showing char- The fungi cultivation was based on PDA medium and an acteristic disease symptoms in the years 2004–2006 (Zi- inoculum of the studied fungus was inoculated with it. The mowska 2008a). inoculum material consisted of rings of 5 mm in diameter, Due to few subject-related papers, which are con- cut out from a 20–day-old maternal colony and cultivated in a dispersed light on the PDA medium at a temperature cerned mainly with the occurrence of P. sclareae on the ◦ of 24 C(Lacicowa 1970). Each isolate was tested four times. sage organs (Subbiah et al. 1996; Voltolino 2001), this The observation of the linear growth of the studied isolates paper presents the undertaken research on the morphol- was conducted for 14 days and the creation of morphologi- ogy of chosen isolates of P. sclareae, as well as the de- cal structures lasted to the 42nd day of the cultivation (Zi- termination of optimal temperature conditions for the mowska 2002). The diameter of the colony was measured most intensive growth and development of infectious every second day. The results of the experiment were sub- material in vitro. jected to a statistical analysis applying a two-way analysis of variance (ANOVA) prepared according to SAS programme. Simultaneously, the macroscopic features of the colony were Material and methods observed. The colour of averse and reverse, the nature of growth of the colony margins and the nature of growth of The studies conducted in the years 2004–2006 involved three the aerial mycelium were determined. Moreover, the shape plantations of sage in the second year of its cultivation, lo- of pycnidia, the type of pycnidia ostioles, the colour of pycni- cated in the districts of Diecinin and Suchodoly in south- dia, the colour of conidial exudate as well as the structure of eastern Poland. The percentage of common sage with symp- pycnidia wall were studied. The measurements of 250 coni- toms of necrosis on the stems and leaves was determined dia α and of 250 conidia β (5 isolates × 50 conidia α and each year at the beginning of and at full vegetation, directly β) and of 250 pycnidia were taken. in the field. The occurrence of fungi was established on the basis of etiological symptoms found on the affected plant organs and on the basis of the mycological analysis conducted Results with the method of artificial cultures. Fungi were isolated from the roots, stem base and leaves disinfected on the sur- Thorough observation of disease symptoms as well as face with the use of the culture mineral medium (Zimowska the mycological analysis indicated the presence of P.

c 2010 Institute of Botany, Slovak Academy of Sciences 604 B. Zimowska

Fig. 2. Twenty eight-day-old colony of Phomopsis sclareae (photo B. Zimowska).

Fig. 1a. Necrosis and cracks in the bark stems from which Pho- mopsis sclareae was isolated. spots and peeling off and breaking of the bark (Fig. 1). In the each year of the studies, these symptoms were observed on the stems at the height of 10 to 18 cm. The percentage of plants with symptoms described above ranged from 10 to 20%. Moreover, conidia, characteristic of Phomopsis species, were found in the cracks of the stem bark. In the stems examined at the beginning of the vegetation there were mostly conidia α, while at full vegetation, conidia β were present as well. In total, 89 fungus isolates were obtained from sage stems as a result of the mycological analysis. A total of 28 isolates came from plants examined at the beginning of the vegetation, which fell towards the end of May and 61 isolates came from the plants examined at full vegetation, which fell towards the end of July (Table 1). Depending on the date of studies, the proportion of P. sclareae isolates within the total fungi obtained from sage stems ranged from 6.6% to 10.5% (Table 1). The in vitro studies indicated a great diversity in the growth of P. sclareae colony cultivated at various temperatures. At a temperature of −6 ◦Cnoneofthe studied fungus isolates produced an aerial mycelium. After the transmission of cultures from a temperature of 6 ◦Cto24◦C, the fungus isolates produced a white- cream aerial mycelium after four days. Single pycnidia appeared after 16 days, whereas conidia were not present until the 42nd day of cultivation. At a temperature of 32 ◦C the colony’s growth was noticed already Fig. 1b. Necrosis and cracks in the bark stems from which Pho- on the second day of the incubation. At first, the aerial mopsis sclareae was isolated (photo B. Zimowska). mycelium was of a white-cream colour and had a tight, velveteen structure. With time, the colour changed into dark brown-grey and a 3-milimeter-long margin of sclareae on the studied plants of sage. From the studied white hyphae was only present on the marginal parts of organs, the fungus was isolated only from the stems of the colony. The colour of the reverse was similar to the sage showing symptoms in the form of oblong, necrotic colour of the averse. At a temperature of 32 ◦Cnoneof Characteristics of Phomopsis sclareae obtained from sage (Salvia officinalis) 605

Table 1. Participation of Phomopsis sclareae isolates in fungal communities obtained from diseased stems of sage in 2004–2006.

Number of isolates (%) Fungi Total beginning of vegetation full vegetation

P. sclareae 28 (6.6%) 61 (10.5%) 89 (8.7%) Other species 386 530 916 Total 424 581 1005

Table 2. The eﬀect of temperature on the diameter of 14–day-old colonie of Phomopsis sclareae on PDA medium.

Temperature 32 ◦C28◦C24◦C20◦C16◦C10◦C5◦C −6 ◦C

Isolates xp1 p2 xp1 p2 xp1 p2 xp1 p2 xp1 p2 xp1 p2 xp1 p2 xp1 p2

S 632 36.0 c B 90.0 a A 90.0 a A 90.0 a A 90.0 a A 78.5 b A 38.5 c A 5.0 d A S 1107 36.8 c AB 90.0 a A 90.0 a A 90.0 a A 90.0 a A 75.0 b A 34.0 c A 5.0 d A S 584 42.0 c AB 90.0 a A 90.0 a A 90.0 a A 90.0 a A 79.5 b A 32.3 d A 5.0 e A S 792 46.0 c A 90.0 a A 90.0 a A 90.0 a A 90.0 a A 76.8 b A 33.3 d A 5.0 e A S 1102 38.8 c AB 90.0 a A 90.0 a A 90.0 a A 90.0 a A 76.3 b A 32.5 c A 5.0 d A

NIR0,05=8.5434 x – diameter of colonies in mm; p1 – differences depending on temperature for isolete – small letters; p2 – differences between isolates at given temperature – capital letters. The means differ in a significant way if they are not marked with the same letter.

Fig. 3. Pycnidia of Phomopsis sclareae SEM ×250 (photo M. Wróbel). the isolates produced pycnidia.Furthermore,after10 and light, ﬂocculent and looser structure. The margin of days of growth, dark swellings of the hyphae appeared. the colony was dug in lobes and the colour of the reverse At a temperature ranging from 20 ◦Cto28◦C, dynamic was similar to the averse. The occurrence of pycnidia growth of the colony was noticed already on the sec- at a temperature of 28 ◦Cand24◦C was noticed already ond day of cultivation (Fig. 2). All of the isolates pro- after 6 days, while at a temperature of 20 ◦Cafter10 duced the aerial mycelium, characteristic of Phomopsis days of cultivation. After 30 days, conidia were present species, in a cream-beige colour, which darkened with in pycnidia; in addition, mainly conidia α were visible time, taking a dark brown-grey colour. Characteristic and conidia β appeared in a greater amount in the 40- zoning of the colony appeared as well, in the creation day-old colonies. At temperatures of 10◦Cand16◦C, of rings of darker mycelium with compact construction after 4 days of growth, and at a temperature of 5 ◦Conly 606 B. Zimowska

Table 3. The size of conidia and pycnidia of Phomopsis sclareae (mean for 5 isolates).

Conidia Size in µm Pycnidia αβ

Own studies 5.5–11.5 × 2.7–4.2 12.5–21 ×1–1.8 250 × 300 F.A. Uecker (1988) 6–12 × 2.5–4 12–20 × 1–1.5 no information

Fig. 4a. Conidia α Phomopsis sclareae ×500 (photo E. Zalewska).

Fig. 4b. Conidia α Phomopsis sclareae SEM ×5000 (photo M. Wróbel).

after 8 days of growth, there were visible loose hypha produced pycnidia until the end of the experiment. of the aerial mycelium around the inoculum, whereas It was statistically proven that the diameter of 14- the typical growth of the colony was observed after 10 day-old colonies of isolates of P. sclareae growing at days of cultivation. At temperatures of 10 ◦Cand16◦C the same temperature did not vary substantially, ex- the fungus isolates produced single pycnidia after 20 cept for the isolate S 632 (Table 2). The studied fungus days of cultivation, whereas few conidia appeared after isolates reached the biggest, maximum diameter at tem- 38 days. At a temperature of 5 ◦C none of the isolates peratures of 28 ◦C, 24 ◦C, 20 ◦Cand16◦Canditvaried Characteristics of Phomopsis sclareae obtained from sage (Salvia oﬃcinalis) 607

Fig. 5. Conidia β Phomopsis sclareae SEM ×4600 (photo M. Wróbel). substantially from the diameter of the isolates’ colonies & Ehrig 2000). In France, P. lavendulae is described as cultivated at temperatures of 32 ◦C, 10 ◦Cand5◦C(Ta- a commonly occurring lavender pathogen (Beus 2005). ble 2). Besides the above mentioned symptoms, fungi from The isolates of P. sclareae produced the most py- Phomopsis genus can cause blights and canceration of cnidia at a temperature of 28 ◦Cand24◦C. They ap- sprouts, rots and mummifications of fruits, stippen of peared irregularly in the darker part of the mycelium, above ground plant organs and in few cases also wilting mostly individually, and they were slightly plunged of plants (Sutton 1980; Uecker 1988). The most charac- into the medium. The pycnidia were circular, dark and teristic symptoms are thought to be those on herba- thick-walled. Numerous mycelial outgrowths were no- ceous and lignified stems (Uecker 1988). Besides, as ticed on the surface of the wall (Fig. 3). The pycni- typical pathogens with a facultative character of par- dia ostioles did not have a neck or a nodule, whereas asitism, they infect plants through natural openings, the mycelial outgrowths present on the surface sur- young undamaged tissues and most frequently through rounded the ostioles in a hardly visible way (Fig. 3). fresh wounds (Machowicz-Stefniak & Kuropatwa 1993). Thick, creamy drops of exudate containing pycnid- Isolating P. sclareae cultures in the present studies, es- iospores came out of the pycnidia’s ostioles. The size of pecially from plant stems at full vegetation, confirms the pycnidia was on average 250 × 300 µm(Table3). the above mentioned information, as plants regener- Conidia α were single-celled, colourless, elliptic in the ation in the form of cutting the stems to the height size range of 5.5–11.5 µm × 2.7–4.2 µm (Fig. 4). Coni- of 8–10 cm was conducted on the plantations in the dia β were filamentous, stick-like bended, long, in the early spring. The injuries made in such a way prob- size range of 12.5–21 µm × 1–1.8 µm(Table3,Fig.5). ably became the gate for penetration of the fungus spores and consequently the development of the dis- Discussion ease. It is known that after infection, the fungus hyphae develop intercellularly in external tissues pene- Isolation of P. sclareae from the stems showing disease trating them into the depths (Pine 1958). Disease symp- symptoms in the form of necrotic spots and bark peel- toms on the stems usually appear after a few weeks and ing off and breaking, as well as the presence of etio- sometimes months (Machowicz-Stefaniak & Kuropatwa logical symptoms in the form of fungus conidia sug- 1993). Conidia α are significantly responsible for infect- gest that these symptoms might have been the conse- ing plants, whereas conidia β are not capable of infect- quence of tissues colonizing the above mentioned fungus ing plants and they sprout only in provocative condi- species. The correctness of this hypothesis is confirmed tions (Uecker 1988). The present research showed that by other literary sources, mentioning the occurrence of both conidia α and β occur in the cracks of the bark species which are pathogenic to herb plants and be- of infected plants. The presence of conidia β mainly on long to fungi from the Phomopsis genus (Uecker 1988; the stems at full vegetation may indicate their role in Gabler & Ehrig 2000; Laine 2003). P. subordinaria is fungus wintering in lignified sage stems (Król 2005). known for causing necrosis and dying of the Plantago The in vitro studies on the morphology of pycnidia laceolata’s flower cluster (Laine 2003). In Germany, P. and conidia, as well as on the character of the growth diachenii causes dying out of caraway umbel (Gabler of the colony, allowed to qualify the studied isolates as 608 B. Zimowska

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