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Isolates and inoculum production
The three isolates used throughout the study were selected from a previous investigation where six isolates were inoculated into 180 1-year-old P. radiata plants (30 per isolate) and where it was shown that they were all pathogenic and did not differ in their relative levels of aggressiveness (R. Ahumada, unpublished). One isolate (CMW33986) of P. pinifolia was used in the first experiment with mycelial plugs and three isolates (CMW33983, CMW33986 and CMW34012) in the second experiment using suspensions of sporangia and zoospores. All the isolates were obtained from needles of P. radiata with the resinous bands characteristic of infection by P. pinifolia. The three isolates selected for the inoculations were chosen because they were similar in relative aggressiveness in a previous study (R. Ahumada, unpublished). Isolates were identified based on culture morphology on the selective medium CARP (Hansen & Hamm 1996) and using the species-specific primers developed by Durán et al. (2009). Emerging colonies were transferred to Carrot Agar (CA) (Erwin & Ribeiro 1996) and maintained between 18–22 °C for 20 days until they could be identified. All three isolates have been maintained in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa.
For inoculum production, 20 replicates of each isolate were grown in V8 agar at 22 °C for two weeks. From the edges of actively growing cultures, 5 discs (7 mm diam.) were transferred to a 60 mm Petri dish containing 25 ml of 10 % V8 broth and incubated for 24 h at 22 °C (Erwin & Ribeiro 1996). The agar discs were washed twice with autoclaved cold distilled water, and then immersed in filtered pond water for 48 h at 22 °C under continuous cool white fluorescent light (6 600 lux). Isolates were examined for the presence of sporangia and those bearing these structures were chilled at 4 °C for 2 h to induce the release of zoospores. The primary zoospore suspension was poured into a sterile beaker and maintained at 4 °C until inoculation. Three aliquots of 10 μl were taken from the beaker and used to determine the zoospore concentration using a hematocytometer. The final zoospore suspension was prepared by adding autoclaved distilled water and adjusting this to approximately 5 x 104 zoospores ml-1. The zoospore suspension was maintained at 4 °C and transported to the field for inoculation on the same day. To determine the viability of the zoospores, three aliquots of 30 μl of the suspension were sampled in the field after the inoculation and spread onto CARP, transported to the laboratory at 4 °C, incubated at 22 °C for two weeks and evaluated for growth.
Inoculation experiments
The inoculations were performed in two different experiments, under laboratory conditions and in the field. The laboratory trials were conducted at Bioforest research facilities in Concepción. The screening facility was set to provide a photoperiod of 12 h of artificial light, at 75 % relative humidity, a temperature of between 18–22 °C and the containers were irrigated daily for 1 h.
The field experiment was installed on the Llico farm (37° 22′ S; 73° 58′ W; Arauco province). A shade house (70 % shade netting) was erected for this inoculation study and a fogging system was installed and applied for 15 minutes, three times per day (10, 14 and 18 hours) to maintain a high level of humidity over the foliage of the inoculated trees. The shade house was surrounded by a plantation of Eucalyptus globulus trees so as to reduce any chance of natural infection by P. pinifolia.
Inoculation of Pinus spp. with P. pinifolia mycelium plugs
Twenty plants (15-month-old, an average of 22 cm tall and 0.8 cm average diam. at the substrate level) of each of thirteen Pinus spp. including varieties [P. arizonica (PAR), P. durangensis (PDU), P. greggii mix of families (PGR1), P. greggii var australis (PGR2), P. greggii var greggii (PGR3), P. maximinoi (PMA), P. muricata (PMU), P. patula mix of families (PPA1), P. patula var longipedunculata (PPA2), P. patula var patula (PPA3), P. pinaster (PPI), P. radiata (PRA) and P. taeda (PTA)] were established in 140 cc containers. The plants were acclimatized for two weeks prior to inoculation in the screening facilities using the conditions described above. Small bark discs (4 mm diam.) were removed from succulent tissue of the stems, between 10 to 12 cm from the growing tips and a plug of mycelium (3-week-old culture of P. pinifolia grown on CA) of similar size was placed into the wounds. Discs of clean CA were used as negative controls. Fifteen plants of each species and the varieties were inoculated with the isolate CMW33986 of P. pinifolia and five plants per species were inoculated as controls. Inoculation wounds were covered with Parafilm to reduce desiccation and contamination. Four weeks after inoculation, the Parafilm and the bark around the inoculation points was removed with a sterile scalpel and the lesion lengths were recorded.
Re–isolation was attempted from the leading edges (top and bottom) of the lesions on all inoculated plants. Small pieces (< 5 mm2) of succulent infected tissue from the leading edges of the lesions were plated onto CARP medium to re–isolate the inoculated organism and ensure that it was associated with the lesions. The identity of 10 % of the resulting cultures that were characteristic of P. pinifolia was confirmed using the PCR specific primers (Durán et al. 2009). Analysis of data was conducted separately for all Pinus species, using the linear model of analysis of variance (ANOVA) and means were separated based on LSD (Least Significant Difference) using the software Statistica V9 for Windows (StatSoft 2004).
Sampling and fungal isolates
A total of 71 isolates of F. circinatum were collected from P. radiata hedge plants, as well as cuttings and seedlings displaying symptoms of infection. The sources of isolates included plant material with typical foliage wilting and resin accumulation at the bases of plants, with depression or abnormal growth in the resin soaked areas. When the bark was removed, diseased tissue could normally be identified as brown or darkly stained (Figure 1). Isolates were obtained from three different nurseries belonging to the Arauco forestry company, namely Quivolgo (31 isolates), Las Cruces (30 isolates) and Los Castaños (10 isolates). The three nurseries were selected because they produce P. radiata plants, they were known to be infected by F. circinatum and they represented the major geographic regions where this pine species is propagated. One nursery was located in Constitución (35° 19’ S, 72° 24’ W), which represents the northern distribution of the F. circinatum; another in Coronel (36° 55′ S, 73° 9′ W) and at the centre of the distribution of the pathogen and the third nursery was near Valdivia (39° 43′ S, 73° 6′ W) in the southern part of the range of distribution of the pathogen in Chile (Figure 2). All the isolates used in this study have been maintained in the culture collection of Bioforest and were selected to represent isolations made between 2002 and 2012. A duplicate set of isolates has been deposited in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria.
Primary isolations from the symptomatic tissue were made by placing a small piece of pitch-soaked pine tissue on Fusarium selective medium (Nash & Snyder 1964). Cultures were incubated at 18–22 °C under normal day light period. The plates were inspected regularly for fungal growth and all the colonies with typical Fusarium morphology were transferred to half-strength potato dextrose agar (PDA) (Merck, Germany). Single conidial cultures were made and stored at –20 °C on sterile filter paper.
Chapter 1
Pinus radiata and its most important diseases, with special reference to Chile
Chapter 2
Pathogenicity and sporulation of Phytophthora pinifolia on Pinus radiata in Chile
Chapter 3
Potential of Phytophthora pinifolia to spread via sawn green lumber: A preliminary investigation
Chapter 4
Phytophthora pinifolia: the cause of Daño Foliar del Pino on Pinus radiata in Chile
Chapter 5
Genetic diversity and population biology of Fusarium circinatum in Chile
Chapter 6
Molecular identification, incidence and pathogenicity of flute canker on Pinus radiata in Chile
Summary
