1.1. Field disease detection
According to several disease reports (Chen et al. 1972; Dafalla and Cousin 1988;
Marcone et al. 2004), the characteristic BGWL phytoplasmal (‘Ca. P. cynodontis’) infected symptoms on bermudagrass (Cynodon dactylon) includes extensively chlorosis (white leaf), small leaf, shortened internode, and axillary shoot proliferation (witches’-broom). The white leaf symptom makes the diseased plants easy to be discovered and distinguished from the green healthy plants. The BGWL diseased plant found at several places, including the GY2015 isolate collected for whole genome sequencing, show the symptoms list above (Fig.
2). Apart from the strain GY2015 collected from Guanyin (Fig. 2A), BGWL diseased plants were also found on sidewalk in Academia Sinica (中研院大門口停車場人行道) (Fig. 2B), the lawn next to Drunken Moon Lake (醉月湖旁草地) in NTU (Fig. 2C), the lawn beside agriculture exhibition hall in NTU (農業陳列館旁草地) (data not shown), the lawn in front of the main library in NTU (振興草坪) (Fig. 2D), the lawn on the green and the path in North Bay Golf & Country Club (北海高爾夫球場的果嶺草皮及小徑旁的雜草) (Fig. 2E, 2F), the lawn on the green in Taipei Golf Club (台北球場的果嶺草皮) (Fig. 2G), the meadow at Huajiang Wild Duck Nature Park (華江雁鴨自然公園) (Fig. 2H), and the lawn at Dulan Cape Café in Taitung ( 台東都蘭海角咖啡草坪 ) (Fig. 2I). For diseased samples from Guanyin beach, NTU main library, and two golf courses are confirmed BGWL phytoplasma infection by 16S rDNA PCR (see below for more information). Other diseased plants showing BGWL symptoms were only suspected to be infected by BGWL phytoplasma
without any molecular biology confirmation. The diseased and healthy plant shown in Fig. 2J were harvest from the same place as in Fig. 2D. The diseased plants showed white leaf and shortened internodes symptom comparing to the healthy plant. Also, the leaves of the diseased plant are more likely to exhibit symptoms of necrosis or dieback compared to healthy plants. The white leaf symptom is occurring from the shoot tip, the upper and younger leaves, to the lower and older leaves. The cuticle covering the epidermis of leaves appears to be lost on the newly emerged white leaves, which may result in lower resistant to light, heat, and drought stress. The white leaves (actually light yellow-green) of the diseased plant, having a greater reflectance at wavelength corresponding to yellow color compared to healthy plant, may be more attractive to insect vector for feeding or laying eggs (Orlovskis 2015). The thinner surface cuticle and juicier leaves could also facilitate the sap feeding insect for haustorium injection. To test these hypotheses, further experiments on leaf surface microscopy observation, leaf wavelength reflectance test, and insect vector behavior studies would be required.
1.2. Microscopy observation on chloroplast morphology
Given the chlorosis symptom of BGWL, we can surmise that the chloroplast of the diseased plant might be confronted with some damages. Therefore, we expect to detect chloroplast morphology changes by microscopy observation. The fresh leaf sample of bermudagrass collected from the lawn in front of the main library in NTU was used for confocal microscopy observation. The diseased plant was confirmed BGWL phytoplasma infection by 16S rDNA PCR (see below for more information). Shown in Fig. 3 is the horizontal view of the flattened leaves without any fixation, staining or dissection. The most obvious difference between healthy and diseased plant is the amount and density of the chloroplast autofluorescence (the red dots). Bermudagrass is a C4 species that has larger and more chloroplasts in the bundle sheath surrounding by the vascular than in the mesophyll
cells. The red chloroplast autofluorescence in the bundle sheath of the white leaf is sparser and weaker comparing to the green healthy leaf, and could barely be seen in the mesophyll area. This indicates that the chloroplast in the diseased plant must be damaged and influenced on both its functionality and quantity. However, there are some unresolved questions: What were the defects of these chloroplasts? Did the chloroplasts have dysplasia issue? Did they retrograde to proplastid? Had they suffered the nutrient lacking stress then turned into senescent chloroplasts? Had they been destroyed by programming death or by other factors such as effector function? To know the answer, we further investigated the morphology of chloroplast by observing the ultra-thin slice sample.
For optical microscopy observation, the sliced and staining (UA in MeOH) leaf vein longitudinal section sample, the same using for TEM observation, was used. In Fig. 4, the bundle sheath cells of the healthy plant are full of chloroplast (50~100% of the volume). In contrast, the chloroplasts were smaller and fewer (<30% of the volume) in the diseased samples. The chloroplast in the mesophyll also has a higher amount in the healthy leaf than in the diseased leave, which is consistent with the confocal observation result. We have not detected any visible difference other than the chloroplast between healthy and diseased sample under optical microscopy observation.
To take a closer look to the morphology of the chloroplast, we observed the leaf vein sample using TEM (Fig. 5). Under the TEM observation, the healthy plant has more and plump chloroplasts in the bundle sheath cells with smaller starch granules or none. The chloroplast showed crescent or spindle shape. The bundle sheath cells are stuffed up by the chloroplasts. On the contrary, comparing to the healthy sample, the diseased sample has leaner, smaller, and odd shaped chloroplast (i.e., hook-shaped or donut-shaped due to the large bending angle, not smooth surface), with larger starch granules. The total chloroplast volume in the bundle sheath cell of the diseased sample is much smaller than in the healthy
sample (most of the space were occupied by a giant vacuole). We also counted the chloroplast number in each bundle sheath cell (Fig. 6). For healthy plant, there are approximately seven chloroplasts in one bundle sheath cell, while in diseased sample there are only five (22 and 23 cells were counted, the P value in t-test is < 0.001). At the same time, we found many irregular shaped chloroplasts with many large starch granules inside and very few (or invisible) thylakoids, which are possibly the senescing chloroplasts. The numbers of starch granules per chloroplast between healthy and diseased plant has no significant difference (data not shown). According to these observations, we hypothesize that the chloroplasts in the disease plant are undergoing a senescing process, because we did not find any proplastid presenting in the bundle sheath. This could be proved if we could observe the chloroplast during the whole process from the beginning of the infection. However, a main challenge for such experiment is the controlled infection system of healthy bermudagrass, which involves rearing and infection of suitable insect vectors.
1.3. Chlorophyll fluorescence and content measurement
To connect the chlorosis symptom with the physiological function of chlorophyll, we quantified the chlorophyll fluorescence and content. The plant samples used for confocal microscopy observation were also used here (different leaves from the same plant). Shown in Fig. 7A and 7B are chlorophyll fluorescence for healthy and diseased leaf samples. Fo is the minimal fluorescence in dark, Fm is the maximum fluorescence in dark, and Fv＝Fm-Fo. The Fm and Fo values from each of four diseased plants are all lower than the values from each of two healthy plants (Fig. 7A). The average of Fv of healthy plants is almost twice as high as the average of Fv of diseased plants (Fig. 7B), indicating that the potential yield from photosynthesis in diseased plant is much lower than in healthy plant. However, the estimated maximum quantum yields (Fv/Fm) of the photochemistry from photosystem II (PSII) for healthy and diseased samples are both around 0.8 with no significant difference, suggesting
that although the potential yield is lower, the efficiency of PSII in diseased plant doesn’t decrease at a serious level. In addition, we also measure the chlorophyll content (chlorophyll A and B) using the same plant sample. Shown in Fig. 7C is the chloroplast content measurement result. Not surprisingly, the chloroplast content in diseased plant is ten times lower than in healthy plant, appearing white leaf symptom of diseased plant.
1.4. Confirmation of phytoplasma by 16S rDNA PCR
The P1/P7 phytoplasma 16S region specific primers were used for diseased samples confirmation by PCR. We got one single band, with approximately 1.8 kb in size, from all BGWL samples (Fig. 8), while no signal was detected in any of the healthy plant samples (data not shown). The PCR product were then sequenced and used for BLASTn search (see materials and methods for more information). The top 10 hits of the BLAST result are all BGWL associated phytoplasmas. For sample GY2015, there are only six bases mismatch and no gap out of 1,723 bp (99% identities for GY2015 and all the other diseased samples) comparing to the reference sequence BGWL-C1 (accession number: AJ550984, Italian strain).
The 16S region of all disease sample contains the BGWL unique signature sequence (5′-AATTAGAAGGCATCTTTTAAT-3′) that mentioned in Marcone et al. 2004. Therefore, the phytoplasma species in the diseased plant samples from Guanyin beach, NTU main library, and two golf courses were all confirmed as ‘Ca. P. cynodontis’.
1.5. Confirmation of phytoplasma by TEM observation
The leaf midvein samples of both green and white leaves from Guanyin beach were used for TEM observation. As shown in Fig. 9, large amount of phytoplasma cells in the sieve element were detected, consistent with the previous study (Dafalla and Cousin 1988).
The phytoplasma cell has one thin layer cell membrane with no cell wall. The DNA filament in the cell is clear to see. The cell shape and size are various. Because of their amorphous
property, phytoplasma cells could pass through the sieve plate by squeezing through the sieve plate pores (the enlarge image in Fig. 9). Most of the phytoplasma cells were assembled near by the sieve plates, while some of the sieve elements were found to be crammed with phytoplasma cells. This would result in a bad conveyance quality of the nutrient by phloem because the nutrient in the sieve element were utilized by the huge amount of phytoplasmas and also because of the blockage of phloem flow by phytoplasma cells. The shortage of nutrient in the phloem would end up forcing the insect vector to stop feeding and leave the disease plant, then they will move on to other new healthy plant host to feed and spread the phytoplasma that they have carried inside their salivary glands. As a result, this promote the dissemination of phytoplasma (Orlovskis et al. 2015).