FUNGI COLONIZING DEAD LEAVES OF HERBS

The material was collected from the Botanical Garden and the Collegium Medicum Medicinal Plant Garden of the Jagiellonian University in Krakow. The investigated species were: lemon balm (Mellisa officinalis L.), common lavender (Lavendula angustifolia Mill.), horsemint (Mentha longifolia L.), sage (Salvia officinalis L.), sweet basil (Ocimum basilicum L.), and wild marjoram (Origanum vulgare L.). The aim of the investigation was to identify fungi causing the death of leaf tissues of herbs from the mint family Lamiaceae. In mycological investigations, 180 fragments of each plant leaves (1,080 dead leaf fragments in total) were placed in a 2% PDA medium. Over 970 colonies of fungi belonging to 48 species were isolated from the dead leaf tissues of the six herb species. Alternaria alternata (toxin-producing), Epicoccum nigrum and Sordaria fimicola were the most frequently isolated. The largest numbers of colonies and species of fungi were isolated from horsemint, while the lowest numbers were from wild marjoram leaves. It was shown that the death of leaves of selected herb species from the Lamiaceae family was caused by various fungi. The results of the mycological analysis confirmed the diversity of species colonizing the leaves of the herbs.


INTRODUCTION
Herbs, like all plants, are susceptible to infectious disease pathogens.Disease symptoms manifested as necrotic tissue are common in herbal plants, despite the anti-fungal properties of active substances derived from this group of plants (G a r i b a l d i et al. 1997; M a c h o w i c z -S t e f a n i a k et al. 2003).Infection can lead to the reduction in the decorative qualities of plants, significant crop loss, and the deterioration of medicinal raw materials.The pathogens most commonly found on herbs are fungi and fungi-like Chromista organisms (M a c h o w i c z -S t e f a n i a k et al. 2003; The aim of this study was to investigate the microorganisms that colonize dead leaves of herbs.

MATERIALS AND METHODS
The research material was collected from the Botanical Garden of the Jagiellonian University and the Medicinal Plants Garden of the Jagiellonian University in Krakow, from May to September 2011 and 2012.The study was conducted on: lemon balm (Mellisa officinalis L.), common lavender (Lavendula angustifolia Mill.) horsemint (Mentha longifolia L.), sage (Salvia officinalis L.), sweet basil (Ocimum basilicum L.), and wild marjoram (Origanum vulgare L.).In mycological investigations, 180 fragments of herb leaves, taken from the edges of living and dead tissue, were placed in a 2% PDA medium.The taxonomic identification of isolated species of fungi was determined using the classifications by B o o t h (1971), E l l i s and E l l i s (1985) as well as D o m s c h et al. (1980).The system by K i r k et al. (2008) for determinate verification in naming fungi according to the Index Fungorum (2012) was used as the basis of classification.

RESULTS AND DISCUSSION
Brownish or brown necrotic spots were visible on the blades and petioles of the tested herb species, sometimes with a narrow lighter border.With time, the leaf tissue dried out and disintegrated.
The mycological analysis of dead tissue covering large areas of leaves showed that these symptoms were caused by fungi from the kingdom Fungi, belonging to 48 species.Over 970 colonies of fungi were isolated from the infected tissue.Most of colonies and species of fungi were isolated from horsemint (205 and 27) and fewest of all from wild marjoram (117 and 10).A comparable number of species of fungi (21)(22) were isolated from lemon balm, lavender, sage and basil, with varying numbers of colonies (135-189).
The toxin-producing species A. alternata was found on all tested herbs, causing the symptoms of alternaria blight on leaves.For basil leaves, this fungus amounted to over 50% of isolates.An equally high proportion of the pathogen was found in the fungal communities isolated from the leaves of wild marjoram, lavender and sage, in which isolates from communities of A. alternata made up over 42, 38 and 30% of isolated fungi, respectively.An even higher proportion of A. alternata (over 76%) was reported by M a c h o w i c z -S t e f a n i a k and Z a l e w s k a (2007) on dying dill plants.The necrotrophs E. nigrum and S. fimicola also occurred frequently on all studied herb species, though their percentage in all communities was lower and more variable.E. nigrum ranged from 7% (in fungal colonies isolated from horsemint leaves) to 24% (from lemon balm leaves).The occurrence of S. fimicola was highest among fungi colonies isolated from leaves of wild marjoram (over 18%) and lowest among fungi found on lavender, horsemint and basil (1-2%).Fungi of the genera Fusarium and Giberella also occurred on dead herb leaves, including F. chlamydosporum var.chlamydosporum on horsemint.The species Mortierella and Umbellopsis (syn.Mortierella) were found on the leaves of all herbs, with the exception of wild marjoram, accounting for over 7% of all isolated colonies.These fungi colonize the youngest, most sensitive leaf blades under conditions of high humidity.Fungi of the species Penicillium and Aspergillus did not colonize dead marjoram leaves, either, but were occasionally found inhabiting other herbs.Fungi of the genera Phoma and Boeremia were found on common lavender, horsemint and sage.The study also detected a small share of fungi from the genus Cladosporium, which has been confirmed by M a c h o w i c z -S t ef a n i a k et al. (2003), M a c h o w i c z -S t e f a n i a k and Z a l e w s k a (2007) as well as Z i m o w s k a (2010).Single Thanatephorus cucumeris colonies were isolated from horsemint, although in the study by Z im o w s k a (2010) the pathogen appeared with a frequency of up to 15% on peppermint and sage.The fungus Trichoderma viride, noted in many studies on herbal crop health, was not found on dead lavender and lemon balm leaves.Septoria melissae, which affects Melissa, was occasionally found on the oldest leaves (Table 1).
The incidence of A. alternata was particularly dangerous on basil leaves, because it led to premature drying and loss of leaves and to a lower quality of raw material, which has been confirmed by M a c h ow i c z -S t e f a n i a k et al. (2003)  Sage leaves were mostly colonized by A. alternata, E. nigrum, Humicola grisea var.grisea, Ilionectria radicicola, Phoma medicaginis and S. fimicola.A comparison of the results to those of Z i m o w s k a (2010) found that the occurrence of the first two species were similar to that on sage plants in plantations and in the collections of the Botanical Garden and the Garden of Medicinal Plants of the Jagiellonian University.The study by Z i m o w s k a (2010) also confirmed the significant role of A. alternata, B. cinerea, E. nigrum and Fusarium and Trichoderma fungi in the death of mint leaves.
According to C h e ł k o w s k i (2012), tissue decay occurs due to the effects of enzymes, or toxins, as well as the effects of necrogenic substances.Mycotoxins produced by Aspergillus and Penicillium inhibit the metabolism of plants and are also produced by pathogens of the genera Alternaria, Phoma and Fusarium, which are characterised by strong phytotoxicity, causing symptoms of necrosis and leaf drop.It can be assumed that A. alternata mycotoxins, or alternariol and tenuazonic acid, played a significant role in causing leaf necrosis in the investigated herb species.C h e łk o w s k i (2012) determined that toxin-producing fungi are generally accompanied by various fungi that are not toxin-producing.This is evidenced by the relatively large number of different species of fungi inhabiting the herb phyllosphere and damaging their leaves.
The present study showed that on the test material, which consisted of selected species of herbs from the Lamiaceae family, leaf death was caused by a complex of fungi and mycological analysis confirmed the diversity of fungi colonizing them.
Z a l e w s k a and M a c h o w i c z -S t e f a n i a k , 2004; Ł a b a n o w s k i et al. 2005; A d a m s k a , 2006; K o ł o d z i e j , 2010; Z i m o w s k a , 2010).
as well as M a z u r and S z c z e p o n e k (2006).The herbal literature also highlights the extensive damage caused by B. cinerea and E. nigrum to basil (G a r i b a l d i et al. 1997; M a z u r and S z c z e p o n e k , 2006).M a c h ow i c z -S t e f a n i a k et al. (2002), Z a l e w s k a and M a c h o w i c z -S t e f a n i a k (2004), M a z u r and S z c z e p o n e k (2006), F r u ż y ń s k a -J ó ź w i a k and A n d r z e j a k (2007) reported the risk of disease caused by Fusarium spp.for herbs.In the present study, only a few colonies of Fusarium spp.were isolated from dead leaves of mint and lavender, and its presence was not confirmed on the leaves of basil, marjoram or sage, while F r u ż y ń s k a -J ó ź w i a k and A n d r z e j a k (2007) reported that lemon balm plants were often colonized by F. avenaceum, F. oxysporum, B. cinerea and S. melissae, peppermint plants byF.avenaceum and  F. oxysporum, and sage by F. culmorum, F. oxysporum  and T. cucumeris.

Table 1
Fungi isolated from dead leaves of herbs