Figure 1.
Schematic map illustrating the spatial distribution of diseased N. attenuata plants in a natural population.
The plant number (n) is indicated for each section of the population (labeled I to XIV). The area of the circles represents the number of plants in each section. The black fractions represent the percentage of diseased plants in each section surveyed on the second monitoring (June 9th 2011).
Figure 2.
Work flow for the isolation of the native fungal pathogen isolates used in this study.
In a native N. attenuata population plants suffered from disease symptoms (chlorosis, curly leaves and dark mycelium-like structures on the abaxial side, illustrated on the left). Leaf samples were taken from diseased plants for fungi isolation. Pathogenicity of the isolates was evaluated using different infection methods. Fungi, which were able to cause severe disease symptoms on plants were re-isolated from infected plant material and further characterized by rDNA sequencing, microscopy and their polar metabolite profiles, resulting in 12 Alternaria spp., 4 Fusarium brachygibbosum, 6 F. solani and one Fusarium sp. isolates.
Figure 3.
Phylogenetic relationship of native fungal pathogen isolates based on their rDNA sequence.
The tree was constructed using the neighbor-joining method based on the sequences of ITS regions plus LSU using MEGA5 software. Sequences were aligned using ClustalW algorithm implemented in the MegAlign software. The numbers on the nodes indicate bootstrap scores in NJ analyses (1000 steps). The number on the scale indicates distance level with relative units.
Figure 4.
Morphology of native fungal pathogens observed by light microscopy.
Conidia of 14 day-old fungal cultures grown for 14 days on PDA in the dark at room temperature. A. Microconidia of F. solani Utah 2. B. Macroconidia of F. solani Utah 4. C. Fungal spores of F. brachygibbosum Utah 4. D. Alternaria-typical conidia. E. Alternaria sp. Utah 10 sporulation clump and conidia. F. Alternaria-typical conidia chain. The white bars represent 20 µm.
Figure 5.
Evaluation of fungal pathogenicity using detached leaf assay.
A. Disease symptoms caused by Alternaria and Fusarium on N. attenuata detached leaves. Agar plugs containing fungus culture were placed on detached leaves of N. attenuata and incubated at high humidity. Detached leaves incubated with pure PDA plugs were used as control. 12 biological replicates were used for each fungal isolate. Photos were taken at 3 and 5 days after inoculation for Fusarium and Alternaria isolates, respectively. B. Necrotic lesion diameters of Fusarium- and Alternaria-inoculated leaves at 5 days after inoculation (dai). The most aggressive isolates (ai) caused the largest necrotic/hypersensitive-like lesion on detached N. attenuata leaves while the moderate isolates (mi) caused visible necrotic/hypersensitive-like lesions only slightly larger than the diameter of pure agar plugs (control).
Figure 6.
Testing different infection setups using F. solani spore suspension and intact N. attenuata plants.
Two different infection methods (spore-spraying and root-dip), three different spore concentration (107, 106 and 105 spores mL−1), two different plant ages (10 and 20 days-old) and two different virulence levels (aggressive and moderate F. solani Utah isolate) were tested with intact soil-grown plants.