New record of Phytophthora root and stem rot of Lavendula angustifolia

Phytophthora cinnamomi was isolated from rotted root and stem parts of lavender as well as from soil taken from containers with diseased plants. Additionally Botrytis cinerea, Fusarium spp. and Sclerotinia sclerotiorum were often isolated from diseased tissues. P. cinnamomi colonised leaves and stem parts of 4 lavender species in laboratory trials and caused stem rot of plants in greenhouse experiments. Cardinal temperature for in vitro growth were about 7,5 and 32°C with optimum 25-27,5°C. The species colonised stem tissues at temperature ranged from 10° to 32°C.


INTRODUCTION
Lavender (Lavendula angustifolia Mill., syn.L.officinalis Chaix) is mainly medicinal and pharmaceutic plant but since the last years it has been grown in ornamental nurseries mainly as small, blooming, hedge culture.The quality of plants may be strongly decreased by Phytophthora species.In 2003 on lavender mother plants grown on greenhouse beds yellowing and browning of single shoots were observed.The disease spread slowly on neighbouring shoots.After the next 6 weeks 3/4 parts of affected plants were destroyed (Fig. 1).On mother plants dark-gray spots on the shoot bases, browning during the next few days and spreading on roots, upwards and on the periphery of affected stems were observed.The disease spread quickly on neighbouring mother plants and cuttings.On plants growing in container -grown nursery brown or dark-brown spots developed on individual shoots spread on roots and upwards.During the next 10 days single or a few yellow-brown to dark-brown shoots on individual plants were seen.The development of symptoms were observed during 2 years before or during flowering of plants.The objective of this investigation was to determine a causal agent of lavender shoot and root rot, development of disease in laboratory and greenhouse trials and relationship between temperature and growth of Phytophthora cinnamomi and colonisation of stem parts of plants.

MATERIALS AND METHODS
Survey of lavender in greenhouse and container -grown nursery.On plants grown in greenhouse shoot rot symptoms were observed only in one year on about 5% of lavender.In container -grown nursery lavender were grown at least 10 years but shoot and root rot symptoms occurred in years 2004 and 2005 on about 10 and 20%,respectively.Diseased plants were collected in plastic bags and transferred to laboratory.The next day plants were removed from pots, substratum were collected in bags whereas plants were washed under tap water and affected shoots were separated, rinsed 3times in distilled water, blotting dried and chosen parts were sterilised over a burner flame.About 5 mm long stem parts were put on PDA medium and plates were checked during 4-day-incubation at 24°C in the dark on the presence of Phytophthora and other genera.Additionally, substratum samples, taken from pots with lavender showing disease symptoms in greenhouse and nursery, were analysed on the presence of Phytophthora spp. in years 2003-2005.Substratum taken from 3-6 pots was mixed about 5 min in plastic bag and half liter of it was put into plastic box, flooded with tap water (about 1 l) and rhododendron leaves cv.Zembla were floated over flooded sample (16 leaf blades/box).Boxes covered with plastic foil were incubated at temperature 22-24°C.After 4-6-day-incubation leaves with necrotic spots were removed, washed with distilled water, blotted dried, disinfected over a burner flame and about 5 mm diameter plugs were transferred on PDA medium.Small parts of colonies, grown around tissues were transferred to PDA slants.Cultures from diseased stems and substratum were grouped on the base of their growth and morphology and chosen, representative isolates were identified to genera and species (S z k u t a 2004).Confirmation of Phytophthora classification to species was performed by DNA analyses using the method described by O r l i k o w s k i et al. (2006).
Colonisation of lavender by Phytophthora cinnamomi.In laboratory trials leaves and stem parts of 4 cultivars of lavender (Tab. 1) were put into plastic boxes on sterilised, wet blotting paper covered with plastic net and inoculated with 3mm plugs taken from the edge of 5-day-old cultures of P. cinnamomi (2 isolates from diseased stem base and substratum) growing on PDA at 24°C in the dark.Boxes were covered with foil and incubated at 22-24°C in the dark.After 5-day-incubation diameter and length of necrosis was measured.In greenhouse trial stem bases of 3 cultivars of lavender were inoculated with 3 mm diam plugs of P. cinnamomi (from diseased stem base) and incubated under covering 12 days.Length of necrosis was measured 8 and 12 days after inoculation.
Relationship between temperature, ranged from 7,5 to 35°C, growth of P. cinnamomi and colonisation of lavender stem parts by the species were studied using the same procedure like in laboratory trials.
Experimental design was completely randomised with 4 replications and 10 leaves, stem parts or plants in each rep.Growth of the species on PDA was estimated on the base of 5 Petri dishes for each temperature.Trials were repeated at least twice.

RESULTS AND DISCUSSION
Isolation of fungi and Algae-like Oomycetes.Similar genera and species were isolated from plants taken from greenhouse and field production (Tab.2).From potential pathogens of lavender Botrytis cinerea (mainly in field), two Fusarium species, Phytophthora cinnamomi and Sclerotinia sclerotiorum were detected from diseased plant parts.P. cinnamomi (Fig. 2) was the most often isolated species both, in greenhouse and field -grown plants.The species was detected from 3/4, 5/7 and 6/7 of analysed plants, respectively (Tab.2).Isolation of Phytophthora cinnamomi from substratum.Using rhododendron leaves as the bait, P. cinnamomi was detected from substratum samples taken from 60 pots with plants showing Phytophthora rot symptoms.Significantly more necrotic spots were observed on rhododendron leaves floated in suspension of substratum collected from diseased lavender grown in greenhouse (Tab.1).
Colonisation of lavender by Phytophthora cinnamomi.Both isolates of P. cinnamomi used for lavender inoculation in laboratory trials, caused necrosis of leaves and stem parts of 4 tested cultivars (Tab.3).On stem parts necrosis spread faster than on leaves.In case of leaf blades inoculation, isolate from substratum colonised tissue slower than that from the stem base.On leaves of cv.Grosso necrosis spread at least 1/3 faster than on 3 other cultivars.On stem parts such differences were not observed (Tab.3).
From diseased plants mainly Phytophthora nicotianae var.parasitica was isolated (Pa p p a s 1978; P u t m a n 1991; M i n u t o et al. 2001a).Growing of plants in Italy under shade and in larger pots reduced disease incidence caused by that species (M i n u t o et al. 2001a).Variable results were obtained with susceptibility of cultivars or local selections toward the pathogen (M i n u t o et al. 2001b).Pa e z et al. (1993) recovered P. palmivora Butler from diseased Lavendula dentata grown in Spanish gardens whereas D a v i n o et al. (2002) isolated the species from rotted root of L. angustifolia in Italy.
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Ta b l e 1Phytophthora cinnamomi isolated from substratum used for lavender growth; mean number of dark-brown spots on rhododendron leaves used as the bait