Environmental Variables 1 Ultraviolet Light

Gadgil and Holden (1976) determined light is an important variable in whether infection proceeds, with higher levels of infection obtained when light intensity was at least 185 W/m2 (400-700 nm) compared with a lower light intensity. Black light tubes produce UV- B (280-315 nm) and UV-A (315-400 nm) light within the solar spectrum, and were used in the developed pathogen assay, in conjunction with visible light to resemble natural sunlight conditions (Section 2.8.2). The assay chamber had to be covered to ensure the mist generated from the water fogger was maintained around inoculated plant material (Section 2.8.5). Therefore, the cover had to allow transmittance of UV-B and UV-A light rays. Possible usable materials were assessed for ability to transmit UV light. The four materials selected for spectrometric analysis were, glad-wrap, petridish plastic, petridish plastic sleeve and parafilm (Section 2.8.2). Figure 4 shows the wavelength each sample recorded the lowest absorbance.

Figure 4 Absorption of light vs wavelength for each sample

0 0.5 1 1.5 2 2.5 3 200 300 400 500 600 700 800 Wavelength (nm) A bs or ba nc e

Gladwrap Lid Plastic Parafilm

Glad-wrap was chosen as the most suitable material, as it had the lowest absorbance around 308 nm, therefore allowing transmittance of UV-B and UV-A light.

3.3.2 Temperature and Humidity

A data logger (Tinytag) was placed inside the assay chamber to determine whether the temperature and relative humidity (RH) within the chamber were similar to the parameters

set for the GMO suite (Section 2.8.3), where the chambers were housed. Table 2. shows the temperature and humidity readings taken from downloaded data from the Tinytag logger, for all pathogen assays which will be discussed in the following sections. Appendix III shows a graphical view of the temperature and humidity data retrieved for the pathogen assay in Section 3.5.4 as an example. As seen in Table 2, the mean RH within the assay chamber was below the constant 80% RH set for the GMO suite, except for trials three, five and seven. The minimum temperature was within ±3°C of the set 16°C (12 h per day). However the maximum temperature fluctuated considerably from the set 24°C for 12 h per day (±8.6°C) for some trials. The average temperature over all trials was within a range conducive to infection (Gadgil, 1974).

Table 2. Temperature and humidity readings for all pathogen assays

Triala _{Relative Min Max Average}

humidity temperature temperature temperature

(%) °C °C °C 1 25.6 16.8 28.7 22.0 2 55.8 16.0 28.3 21.7 3 80.9 14.2 32.6 19.4 4 64.3 13.1 27.6 18.2 5 88.6 13.1 27.6 18.1 6 45.8 13.7 27.6 18.2 7 88.6 13.1 27.6 18.1

a _{1, mycelium vs conidia; 2, seedlings vs cuttings; 3, milli-q, pH 3, rainwater; 4, DSM, pH 5, PNA; 5,}

milli-q, milli-q + Y, seed broth, lowDB; 6, double inoculum; 7, pks-_.

Note: The relative humidity percentage represents the average from data recorded per pathogen trial.

3.3.3 Water Source

Mineral deposits on pine needles watered with laboratory tap water were discovered by a previous researcher in this laboratory (West, 2004). As the buildup of mineral deposits increases over a period of time, it may be inhibitory to dothistroma infection, perhaps by blocking access to pine needle stomata. Figure 5 is an image of mineral deposits on the surface of a pine needle taken from an assay chamber which contained laboratory tap water. Generally the buildup of minerals on the needle surface is clearly visible to the naked eye and does not require microscopic analysis.

Figure 5 Mineral deposits on pine needle

Pine needle viewed at 100x magnification

The use of RO water (distilled water) instead of tap water reduced the buildup of mineral deposits on the surface of pine needles (visual observation).

3.3.4 Scoring Infection

The procedure outlined in Section 2.8.6 for scoring disease was followed for all subsequent pathogen assays, to ensure all data collections were consistent between assays conducted at different times. Chlorotic symptoms of disease, such as areas of brown/tan colouration within regions of the pine needle maybe due to other organisms or tissue damage not necessarily associated with D. septosporum. Therefore, more specific signs of D. septosporum infection (red bands and fruiting bodies) were used to evaluate the success of the pathogen assay.

All needles with red-bands were viewed at 40x magnification to determine the presence or absence of fruiting bodies. Under normal white light of the dissecting microscope or in natural daylight, the bands display a red colouration that is distinct from normal chlorotic lesions often seen on needles, appearing tan or brown. The red colouration is due to the red pigmented mycotoxin, dothistromin. In Figure 6, photograph A shows multiple red bands on single needles (arrow heads), while photograph B shows a mature fruiting body erupting through the epidermal layer of the needle tissue, both signs characteristic of dothistroma disease.

Figure 6 Signs and symptoms of D. septosporum disease

Photograph B taken at 100x magnification

The fruiting body is oval in shape, and splits the epidermal layer longitudinally. Sometimes fruiting bodies appeared in areas that did not show the characteristic red band. However on several occasions, red crystals were visible around the outside and within the fruiting body. It is probable these were dothistromin crystals.

Figure 7 shows a typical comparison between an infected seedling (A) and uninfected seedling (B) within the pathogen assay system. The majority of infection usually occurred on the lower half of the seedling, with the young needles becoming necrotic while still attached to the seedling. The red bands are prominent and distinguishable along the length of the pine needle (arrow heads). In instances where a chlorotic region within a green needle is present, and this region is not prominently reddish in colouration (characteristic red-band), the needle is classified as chlorotic and subsequently classed as non-infected. In addition, a small number of the lower needles on the pine seedling become necrotic due to natural reasons (arrow photograph B). These needles are very distinct from infected needles as they do not display the banding pattern and are generally of a dark brown colouration, as opposed to the tan colouration of chlorotic needles or red colouration of infected needles.

A

Figure 7 Infected and uninfected pine seedlings

A = Dothistroma infected seedling, B = Uninfected control seedling

3.3.5 Natural Inoculum Validates Developed Pathogen Assay System

Once an adequate assay chamber had been developed which met all required environmental conditions for infection to occur, ie. light, temperature and humidity, the next step was to use natural inoculum in order to test the system. This was a preliminary test prior to the set up of the pathogen assays with cultured inoculum.

The assay chamber was set up as described in Section 2.8.1, with infected needles (of the same batch from which NZE10 had been isolated), suspended over pine seedlings. Six weeks later the needles on the top third of the pine seedling showed signs of dothistroma disease, ie. red bands and fruiting bodies. During the experiment the fascicles containing the natural inoculum became very wet due to continuous misting and subsequently drooped down onto the top of the seedlings. Consequently, many needles also became very necrotic, appearing to be swollen and water logged. However, the fruiting bodies within the red bands on diseased needles were very visible. Figure 8 shows the assay chamber set up and also a pine seedling six weeks post inoculation.

1 cm

A

2 cm

B

Figure 8 Natural inoculum pathogen assay

A = Natural inoculum suspended over pine seedlings, B = Infected pine seedling

In Figure 8 the arrows in photo A indicate the source of natural inoculum, whilst the arrows in photo B indicate necrotic needles which upon closer observation showed signs of dothistroma disease.