Centre of Competence for the Innovation in the Agro-Environmental Field, AGROINNOVA, University of Torino, Grugliasco, TO, Italy Molecular Detection of Phytophthora cryptogea on Calendula officinalis and Gerbera jamesonii Artificially Inoculated with Zoospores Yuan uan Li, Daniela Daniela Minerdi inerdi, Angelo Angelo Garibaldi aribaldi and and Maria Lodovica Maria Lodovica Gullino ullino AuthorsÕ address: Centre of Competence for the Innovation in the Agro-Environmental Field, AGROINNOVA, University of Torino, via Leonardo da Vinci, 44, 10095 Grugliasco, TO, Italy (correspondence to Yuan Li. E-mail: liyuan80416@yahoo.it) Received April 15, 2008; accepted August 27, 2008 Keywords: Phytophthora cryptogea zoospores, specific primers, polymerase chain reaction, SYBR Green real-time PCR Abstract In this work, a protocol for zoospores production of Phytophthora cryptogea, an economically important plant pathogen was optimized. Five different concen- trations of zoospores (5 · 10 5 , 5 · 10 4 , 5 · 10 3 , 5 · 10 2 ,5 · 10 1 zoospores ⁄ ml) from four different iso- lates of P. cryptogea (Maria 1, Maria 2, S3 1-A, Amazzone) were used as inoculum on pot marigold (Calendula officinalis) and gerbera (Gerbera jamesonii) plants. Maria 1 was the most virulent isolate both on pot marigold and gerbera plants according to disease severity. A rapid and sensitive pathogen DNA extrac- tion protocol suitable for large quantities of plant sam- ples was adopted. Conventional polymerase chain reaction (PCR) was able to detect the pathogen in arti- ficially inoculated symptomless pot marigold (collected day 12) and gerbera plants (day 8) after pathogen inoculation, with the suspension of 5 · 10 5 ,5 · 10 4 , 5 · 10 3 P. cryptogea zoospores ⁄ ml. Real-time PCR showed the possibility to detect the pathogen in arti- ficially inoculated symptomless pot marigold (collected day 8) and gerbera plants (day 4) after pathogen inoculation, with the suspension of 5 · 10 5 ,5 · 10 4 P. cryptogea zoospores ⁄ ml. The first symptoms appeared on pot marigold plants 14 days after pathogen inocu- lation and on gerbera plants 10 days after inoculation. Real-time PCR showed the possibility to detect the pathogen 4 days before conventional PCR and 6 days before the appearance of disease symptoms both on pot marigold and gerbera plants. Introduction Phytophthora cryptogea is a zoospore-producing chro- mista recognized worldwide as an important pathogen of many crops, including ornamentals, such as Ger- bera jamesonii and Calendula officinalis, particularly those grown under glass or plastic, and many tree crops as well (Erwin and Ribeiro, 1996). The host range for P. cryptogea includes a large number of genera distributed among more than 23 plant families (Erwin and Ribeiro, 1996), being able to cause severe root rot and crop losses (Garibaldi et al., 2003). The pathogen is easily recovered from irrigation water and nutrient solution and soilless growing systems (Garibaldi et al., 2003) where it can survive in the absence of a suitable host (Stanghellini and Rasmus- sen, 1994). The epidemic spread of disease is often based on the rapid dispersal in water from host to host by zoospores that are released from sporangia (Walker and West, 2007). Zoospores are the natural means for P. cryptogea to spread: the quantity of zoospores present at a site is initially dependent on the production of sporangia, which may be affected by various environmental factors (Hong et al., 1999). Zoospores of P. cryptogea are able to survive in for- est soil for at least 14 days, which is a sufficient per- iod to be spread from one area to another (Bumbieris, 1979). The stage of zoospore production is the most susceptible phase of the life cycle of zoo- sporic fungi and at this stage disease control must be efficient (Raftoyannis and Dick, 2006). Therefore, the early, rapid and accurate detection and identification of P. cryptogea in plants is essential in order to reduce their spread and to improve prevention, espe- cially when pathogens have a wide host range (Ehret et al., 2001; Schena et al., 2004). Moreover, quantita- tive analyses are of basic importance if there is a threshold level of pathogen propagules at which chemical or other control measures must be applied (Schena et al., 2004). Traditional identification based on morphological or physiological characteristics is time-consuming, labour-intensive, and requires consid- erable knowledge of the genera and species involved (Grote et al., 2002). Molecular approaches to the sen- sitive identification and detection of P. cryptogea developed in recent years open up new pathways for J Phytopathol 157:438–445 (2009) doi: 10.1111/j.1439-0434.2008.01512.x Journal compilation Ó 2009 Blackwell Verlag, Berlin No claim to original US government works