Robillarda sessilis, a rare coelomycete isolated from Scots pine seedlings

Robillarda sessilis (Sacc.) Sacc. was found during studies of the fungi inhabiting stems of healthy Pinus sylvestris seedlings in a sample nursery in central Belarus. This species is a remarkable fungus with conidia bearing three long hair-like appendages. It was selected as a generic type for Robillarda, an anamorphic genus with unknown teleomorph (Nag Raj 1993). Altogether 35 species were published under this generic epithet, 12 of which were referred, according to Nag Raj (1993), to seven other genera.


INTRODUCTION
Robillarda sessilis (Sacc.)Sacc.was found during studies of the fungi inhabiting stems of healthy Pinus sylvestris seedlings in a sample nursery in central Belarus.This species is a remarkable fungus with conidia bearing three long hair-like appendages.It was selected as a generic type for Robillarda, an anamorphic genus with unknown teleomorph (Nag Raj 1993).Altogether 35 species were published under this generic epithet, 12 of which were referred, according to Nag Raj (1993), to seven other genera.

MATERIAL AND METHODS
The inventory of the fungi was carried out by sampling plants in 1 m 2 plots on a 800 m 2 plantation situated in central Belarus.A first sample (taken 2 VI 2009) included 5 well-developed seedlings from a single plot located in the center of plantation.A second sample (taken 22 VI 2009) included 25 equally well-developed seedlings collected from 5 plots (5 plants per plot).Four plots were situated near corners of the plantation, one plot in the plantation center (this central plot position did not coincide with the plot in previous sampling).
For isolation of the fungi, the seedlings were washed 3 min under a strong stream of tap water on a sieve.Each seedling stem, from the root neck to the foot of leaf whorl, was cut using a sterile razor blade into ca 2 mm long segments.The segments were put on malt extract agar (MEA: 1% malt extract, Amresco, USA, and 1.5% agar) with addition of tetracycline 30 μg/mL (Amresco), and incubated for 10 days at 26°C.For the control that the propagules did not originate from tap water, 1.5 mL of the water was poured in Petri dish with solid 2% MEA, in 3 replicates.Mycelia growing from stem pieces and producing pycnidia, were transferred for storing on MEA slants under mineral oil.For describing cultural morphology, isolates were taken from storage and cultured on MEA (2% malt extract, 1.5% agar) at 26°C in the dark.
For describing micromorphology, preparations were mounted in 3% KOH water solution.Dry cultures were deposited in V.F.Kuprevich Institute of Experimental Botany Herbarium (MSK-F).

RESULTS
It the first sample from the plantation center (2 VI 2009), one isolate of R. sessilis was obtained.In the second sample (22 VI 2009) two isolates were obtained from 2 of 25 studied plants, one from central, one from a corner plot.The morphological diagnosis of R. sessilis, based on cultures, is given below.
Morphological description.After 1 week: mat 25 mm in diam, more or less rounded, colorless, low felty, near 1 mm high, more or less fasciculate or tufted in periphery zone and just near the point of inoculation; margin somewhat wavy, abrupt, with scarce cilia-shaped hyphae; reverse pale apricot yellow.After 2 weeks: mat about 50 mm in diam, very pale pinkish cream, in central and middle zone floccose-felty; periphery zone clearly delimited, ca 10 mm wide, low velvety, with slight radial depressions; margin without aerial growth, transparent; reverse in central and middle zone brownish yellow with orange tint, with blackish or dark olive spots due to the presence of conidiomata.Conidiomata occurring about 1.5-2 weeks post inoculation, especially developing when the medium dries up, in mat periphery and middle zone scattered, but at the border between these zones abundant and often aggregated, forming a ring, having dark brown color in reverse (Fig. 1).
Substrata and general distribution.R. sessilis was documented from quite variable hosts and substratum types: on stems (bark), dead branches, leaves (causing leaf spot), seeds of Bischofia, Cocos, Ficus, Fragaria, Fumana, Ludwigia, Magnolia, Paeonia, Quercus, Randia, Rosa, Rubus, Vitis.The fungus has been recorded in Europe (Hungary, Italy), Asia (India), North America (USA), Caribbeans, Africa (Angola), and on the plant material imported from Japan.The majority of records are from India (Nag Raj 1993).In 1970 this species was described from Northea seeds imported to St-Petersburg, Russia, from Indonesia, under the name Mycohypallage northeae (Melnik 1970).Gasich (1995) collected this fungus in Saratov oblast, Russia, where it caused leaf spot disease of Eryngium (see also Melnik 1997).So far, five localities, including the type locality in Italy and our find, but excluding the find on imported seeds, are know from Europe today.In Nebraska, USA, R. sessilis was collected on Pinus ponderosa, but details about the age of the plants or the kind of organs are unknown (Nag Raj 1993).The fungus was also isolated from soil in Australia (Matsushima 1989) and from a soil sample collected in mountain pine forest in Pakistan (Matsushima 1993).
Our case indicates that R. sessilis is capable of infecting stem tissues of healthy, vigorous Pinus sylvestris seedlings as either an endophyte or phylloplane component.The fungus can be named more widely 'associated with seedling' because of used method of sterilization.Conidiomata were not observed on this host.Only three plants colonized by the fungus, were detected among 30 ones subjected to cultural experiment, and thus the species can be regarded as rare in the studied plantation.