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narum) cv. H 65-7052 expressed severe yellowing. Subsequently, similar symptoms were reported in Florida. (Comstock et al., 1994), Africa (Bailey et al., 1996, ...
Plant Pathology (2008) 57, 178–189

Doi: 10.1111/j.1365-3059.2007.01696.x

Symptom expression of yellow leaf disease in sugarcane cultivars with different degrees of infection by Sugarcane yellow leaf virus

Blackwell Publishing Ltd

A. T. Lehrerab and E. Komora* a

Pflanzenphysiologie, Universität Bayreuth, D-95440 Bayreuth, Germany; and bHawaii Agriculture Research Center, Aiea, HI, 96701 USA

Sugarcane yellow leaf virus (ScYLV) is present in many sugarcane growing areas of the world. It is suspected to cause yellow leaf disease (formerly called YLS, yellow leaf syndrome) of sugarcane. This study investigated symptom expression in a selection of cultivars classified into three groups; ScYLV-susceptible/infected, ScYLV-resistant and intermediately infected cultivars grown in plantation fields in the islands of Hawaii. Incidence of yellow leaf symptoms was correlated, though not tightly, to the presence of ScYLV. The correlation is based on two factors: (i) only ScYLV-infected cultivars (from both susceptible and intermediate groups) showed severe symptom expression, and (ii) ScYLV-infected plants had four times higher symptom incidence than virus-free plants of the same cultivar. The yellow leaf symptom expression fluctuated, peaking at 200, 350, 500 and 600 days after planting. These symptom peaks were correlated with an increase of ScYLV content in the intermediately infected group of cultivars. No nutritional, environmental or field factor could be identified which clearly influenced symptom expression. It is speculated that the symptom expression is elicited by assimilate backup in the stalks and that the fluctuation of symptom expression is caused by the growth rhythm of mature sugarcane stalks. Keywords: Saccharum officinarum, ScYLV susceptible and resistant cultivars, symptomatology

Introduction Yellow leaf syndrome (YLS, now called yellow leaf disease) was first described in Hawaii (Schenck, 1990) when plantation fields of sugarcane (Saccharum officinarum) cv. H 65-7052 expressed severe yellowing. Subsequently, similar symptoms were reported in Florida (Comstock et al., 1994), Africa (Bailey et al., 1996, where older reports had already noticed a ‘yellow wilt’) and Brazil (Vega et al., 1997). In the past ten years YLS has also been reported from several other sugarcane growing areas. A virus was isolated from infected plants which was identified as a member of the Luteoviridae and named Sugarcane yellow leaf virus (ScYLV) (Scagliusi & Lockhart, 2000). However, no strict correlation between the presence of ScYLV and YLS was observed. In some cases, yellow leaf symptoms were seen although ScYLV could not be detected. A phytoplasma was found to be responsible for these symptoms (Cronje & Bailey, 1999). In other cases, no yellow leaf symptoms were seen despite the *E-mail: [email protected] Accepted 30 May 2007

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presence of ScYLV (Comstock et al., 1998), even when the very sensitive RT-PCR technique was used as assay for the virus (Aljanabi et al., 2001). Aljanabi et al. (2001) also found that the coexistence of both ScYLV and sugarcane phytoplasma enhanced the syndrome incidence in Mauritius. Because of the loose correlation between ScYLV and yellow leaf symptoms, cane growers were not convinced that ScYLV was involved. Some researchers even concluded that there was no yellow leaf virus disease. They suggested that the observed symptoms were a pathogen-independent ‘autumn decline’ (Matsuoka & Meneghin, 1999). A survey conducted in Hawaii using a tissue blot immunoassay revealed that in some cultivars all plants were infected. These cultivars were therefore called ScYLVsusceptible. In contrast, some other cultivars appeared completely free of ScYLV and were called ScYLV-resistant (Schenck & Lehrer, 2000). These resistant varieties were never diagnosed with ScYLV even when grown in close proximity to infected cultivars or when infested with viruliferous aphids. The present study compared ScYLVsusceptible, and ScYLV-resistant cultivars as well as cultivars described as intermediately infected in Hawaii with respect to yellow leaf symptom expression. The purpose of the study was to show whether there was any correlation between ScYLV and yellow leaf disease when © 2007 The Authors Journal compilation © 2007 BSPP

Sugarcane yellow leaf and ScYLV infection

symptoms of different cultivars were analysed carefully with respect to symptom severity and the timing of symptom expression. Susceptible plants generated by tissue culture are virus-free (Chatenet et al., 2001; Fitch et al., 2001; Parmessur et al., 2002) and therefore, a comparison of infected plants and virus-free plants of a susceptible cultivar also was conducted to obtain information about the role of ScYLV in yellow leaf symptom expression.

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Test plots were placed within commercial plantation fields and were fertilized and irrigated according to the plantation practice. The test plots in Kunia, Oahu, were treated identically to the field practice of the commercial fields in Maui. Plots in Maunawili, Oahu, were not irrigated due to higher rainfall, but otherwise treated similarly to plantation fields in Maui which also lie at a higher elevation.

Growth of sugarcane plants in sand culture

Materials and methods Cultivars Four commercial cultivars susceptible to and infected by ScYLV were selected: H 65-7052, a high yielding Hawaiian cultivar, in which yellow leaf symptoms had been first observed in 1988; H 73-6110, another high yielding cultivar, which was phased out as a commercial cultivar in 1995 after a widespread yellow leaf outbreak; H 77-4643, which is in commercial use in Kauai; and H 87-4094, a promising new commercial cultivar which was recently eliminated from plantation fields because of yield decline. Four resistant cultivars were selected for study, all of which are used commercially in Maui: H 78-3567, H 78-4153, H 78-7750 and H 87-4319. Two other cultivars were tested which were candidate commercial varieties, H 78-3606, which is infected by ScYLV, and H 82-3569, which is ScYLV-resistant.

Planting and test plot arrangements Seed pieces (setts) of the different cultivars were stem cuttings of 50 cm length, containing three nodes, from seed cane fields in Maui or Kauai. They were incubated in water at 50°C for 30 min and then dipped in fungicide solution (Tilt, Novartis). They were planted following plantation practice in parallel rows 1 m apart with a drip irrigation tube in the centre and covered with soil. Each field plot consisted of 16 setts per cultivar, in two parallel rows 1 m apart and 3 m in length. Eight fields were selected for the test plots, three at the Hawaiian Commercial & Sugar (HC&S) plantation in Maui (field 200, field 602 and field 807), three at the plantation of Gay & Robinson in Kauai (field 330, field Punakaawe 10 and field Moomoku 10) and two fields of the HARC experiment station in Oahu (field Kunia and field Maunawili). All fields with the exception of Maunawili are located in the dry, leeward areas of the Hawaiian Islands. Three test plots were installed in each of the fields and each test plot contained nine or ten cultivars planted in double rows side by side as described above. The order of cultivars within each test plot was randomized to minimize neighbour effects. Nine of the cultivars were replicated three times in each of the eight fields listed. Cultivar H 77-4643 was only placed in the three fields in Kauai (field 330, field Punakaawe 10 and field Moomoku 10). Fields were planted at the end of January in Maui and Oahu and mid June in Kauai. Plant Pathology (2008) 57, 178–189

Experiments were performed with the susceptible cvs H 65-7052, H 73-6110 and H 87-4094 to see whether a nutrient or water deficiency provoked symptoms of yellow leaf. Calcium, phosphate and water shortage have been postulated as possible reasons for symptom development. Seed pieces with only one bud were treated with fungicide and hot water as described above and then germinated on wet paper for 1– 2 weeks until the shoot and roots were about 5 cm in length. They were then planted in pots containing 10 L of wet, washed quartz sand. The plants were grown in a greenhouse at ambient temperature and light conditions. Watering was by drip irrigation. Each treatment was performed on two plants of each cultivar and was carried out for 6 months. For control plants, a nutrient solution developed for Hawaiian sugarcane cultivars was used (Martin & Eckert, 1925; Sholto Douglas, 1956) and contained the following micronutrients: 0·1 mm boric acid, 0·02 mm MnCl2, 0·0015 mm ZnSO4, 0·001 mm CuSO4, 0·001 mm (NH4)6Mo7O24, 0·2 mm NaFe(III)-EDTA, 0·01 mm Na2SiO4. The macronutrients were 4·5 mm CaCl2, 2 mm KNO3, 3·5 mm NH4NO3, 1 mm MgSO4, 0·4 mm (NH4)2HPO4. The nutrients were added to the irrigation water by automatic injectors. Watering occurred three times per day with 1 L nutrient solution per pot. Plants under calcium deficiency had the same nutrients as the control plants, but 4·5 mm NaCl was substituted for calcium chloride. Plants under phosphate deficiency received the same nutrients as the control plants except that 0·4 mm (NH4)2SO4 was substituted for the ammonium phosphate. For the plants under water shortage the micronutrients and the macronutrients had the same composition as for the control plants. The plants were watered three times per day with only 0·5 L of the nutrient solution. The drought experiment was performed with plants that received either the standard nutrient solution, or plants receiving the P- or Ca-deficient nutrient solution. The nutrients were added to the irrigation water by automatic injectors; the amount of nutrients given to the plants was the same as for the plants which were watered normally.

Generation of virus-free susceptible sugarcane plants Virus-free cv. H 87-4049 was generated by meristem tip culture as described in Fitch et al. (2001). The virus-free plants were propagated in a seed cane plot at the Brigham Young University farm in Laie, Oahu. This farm is geographically isolated from any current or recent

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commercial sugarcane fields that could harbor the virus. No ScYLV-infection in this location has been found in over five years.

Generation of ScYLV-free aphids and experimental infestation of plants with aphids Aphids were collected from fields in Oahu, Hawaii, and colonies were maintained on infected sugarcane plants (cv. H 73-6110) in insect-tight cages. When virus-free aphids were needed they were collected with a fine wet brush and transferred onto virus-free H 87-4094 or H 78-4153 plants. Since ScYLV is not passed to aphid offspring, several transfers of offspring on virus-free plants produced virus-free insects. They were then maintained continuously on virus-free plants of cv. H 87-4094 in insect-tight cages. For the controlled aphid infestation experiment 10 aphids were collected from the colonized plants and transferred onto 3-month-old H 65-7052 plants in sand culture. Aphids were maintained for 1 week in clipon cages on the plant and then removed. Plants were maintained for a further 2 months. The aphid feeding trials were performed in the same way with viruliferous Melanaphis sacchari, with virus-free M. sacchari and with Sipha flava aphids.

Determination of viral infection The test plots were inspected at 6–8 week intervals. Three leaf samples from each cultivar in each test plot were collected for tissue prints. The leaves collected were the youngest fully expanded (top visible dew lap) leaves on each plant. The stalks from which the samples were taken had been marked with coloured ribbon. The presence of ScYLV was tested by tissue blot immunoassay as described in Schenck et al. (1997) and Fitch et al. (2001). Tissue prints of the leaf midrib were performed in triplicate on nitrocellulose membranes (Biorad Transblot). After a pretreatment with chloroform to extract chlorophyll, the membranes were blocked with TBS-buffer (50 mm Trizma base plus 50 mm NaCl pH 7·5 containing 2% dry milk), then incubated with rabbit antiserum raised against whole ScYLV. The blots were probed with goat anti-rabbit alkaline phosphatase-conjugated antibody followed by colour development using BCIP/NBT (5-bromo-4-chloroindol-3-yl phosphate/Nitro Blue Tetrazolium) substrate (Sigma). The method visualizes the presence of viral coat protein in the print of the midrib bundles in infected plants. The method is sensitive enough to detect the virus in the phloem of a single bundle. No grading of the intensity of colour was performed, i.e. whenever a clearly positive reaction was observed, the plant was considered as infected irrespective of weak or strong dye development or number of infected phloem bundles.

Screening of plants for yellow leaf disease symptoms The test plots were inspected at 6–8 week intervals. The six top leaves from number –3 to +3 (top visible dew lap

as leaf number +1) were inspected. The overall yellowing of leaves of all plants in each plot was graded and recorded. The rating result indicated the symptom severity which was present in the majority of the plants in the test plot. The yellow leaf symptoms were graded visually on a scale from 0 to 6 according to the following scheme (Fig. 1): 0: no leaf yellowing, 1: small, faint yellowish streaks on a part of the leaf midrib, or slight leaf yellowing, 2: faintly yellow midrib, 3: yellow midrib of source leaves, but leaf blade still green, 4: yellowing of leaf blade parts closely adjacent to the midrib, 5: yellow midrib and yellowish major veins outside the midrib, 6: yellow leaf blade, sometimes already partially dried out leaf edges.

Data analysis The predominant symptom grade was recorded for each cultivar in the three test plots of a field at each inspection event, usually at 6–8 week intervals. The median and quartile range of the symptom grade were calculated for each cultivar at a particular age, giving an impression of the prevalent symptom severity for a cultivar at each age. Since the test fields were very different in climate and soil, the symptom severity could be very variable even at a fixed plant age. To investigate symptom seasonality the symptom expression grade per field was calculated by adding up the symptom grades of a cultivar in all test fields at an inspection event, divided by the number of test fields. In order to summarize symptom expression over the entire growth period and to account for both frequency and severity of symptom expression of a cultivar, a ‘cultivarspecific symptom expression value’ per field and per inspection was calculated. Therefore the grade of the symptom expression in each cultivar observed in all eight test fields and at all inspection events was summarized. This number divided by the number of fields and the number of inspection events gave an estimate of the symptoms exhibited by each cultivar over the entire growth period. Similarly a ‘field-specific symptom expression value’ was calculated, adding up all observed symptom grades in a test field and dividing by the number of cultivars in the field and the number of inspection events.

Results Yellow leaf disease symptoms and yellow leaf diseaselike symptoms Different grades of leaf yellowing were observed during the project. The yellowing started from the midrib and proceeded successively to the leaf blade and eventually led to completely dry leaf edges (Fig. 1). This progression of yellowing was different from yellowing during senescence (caused by age or by nutrient shortage) where the yellowing started from the outer side of leaf blades. However, yellow leaf disease-like leaf yellowing was also observed when major leaf veins were mechanically interrupted, for example when the midrib of a leaf broke because of strong wind or when a partial cut of the stalk occurred. Also massive Plant Pathology (2008) 57, 178–189

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Figure 1 Grading of yellow leaf symptom expression in sugarcane from grade 0 (no symptom) to 6 (complete yellowing with partially dried leaf blade rims). Green leaf top morphology of (a) healthy plant, (b) bunchy leaf top of yellow leaf diseased plant; and (c) and (d) yellow leaf disease-like symptoms caused by the aphid Sipha flava or by a broken leaf midrib respectively.

infestation by Sipha flava, an aphid which does not transmit ScYLV (Schenck & Lehrer, 2000), caused local yellowing at the feeding site (Fig. 1c). Care was taken to distinguish yellow leaf disease symptoms from these other yellowing events during monitoring. However, the strong yellow leafsymptoms (grades 2 – 6) and the ‘bunchy top’ morphology of the green leaf top, when the leaves emerge very closely to each other because of the shortened internodes (Fig. 1b) were not observed after mechanical or insect-caused damage.

Correlation between immunoassay for ScYLV and symptom expression A previous screening of Hawaiian sugarcane cultivars had shown that there were large differences with respect to positive reactions for ScYLV in the plants. Some cultivars always reacted positively to the immunoblot test for ScYLV and were therefore called susceptible and infected. Others almost never reacted positively and were called resistant. Some cultivars were intermediate in their response to the test and showed between 4 and 30% positive reactions, the so called intermediately infected cultivars. Three or four commercial cultivars of each of these susceptibility groups were selected for the present study (Table 1). The selected cultivars were planted in the field plots and inspected at 6–8 week intervals for yellow leaf symptoms over a 21-month growth period. Plant Pathology (2008) 57, 178–189

Table 1 Infection status of selected cultivars of sugarcane with Sugarcane yellow leaf virus. The presence of ScYLV was tested over the whole growth period from 1 to 21 months by tissue blot immunoassay

Cultivar

Presence (%) of ScYLV/number of samples

Cultivar classification

H 65-7052 H 73-6110 H 77-4643 H 78-3567 H 78-3606 H 78-4153 H 78-7750 H 82-3569 H 87-4094 H 87-4319

35/244 (14) 231/245 (94) 21/121 (17) 11/246 (4) 214/234 (91) 2/244 (1) 0/221 (0) 1/246 (0) 212/244 (87) 3/244 (1)

Intermediately infected Susceptible/infected Intermediately infected Intermediately infected Susceptible/infected Resistant Resistant Resistant Susceptible/infected Resistant

The ScYLV-infected cultivars exhibited yellow leaf symptoms more often than the resistant cultivars. Plants with symptoms were observed in 28 cases/susceptible cultivars, but only in 4·7 and 19 cases for resistant and intermediately infected cultivars respectively. The symptom grades in the infected susceptible cultivars reached grade 5, whereas symptoms of the resistant cultivars rarely exceeded grade 1 (Fig. 2). However, the

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with low incidence of symptoms (Fig. 3). No symptoms were observed in the first 100 d of growth. Subsequently symptoms appeared in the two susceptible cultivars (H 65-7052 and H 73-6110), first at a low grade scores around day 200, then at higher values around 300-400 d. Symptoms disappeared at day 400 and reappeared again around days 500 and 600. Cultivar H 78-3606 showed only few grade 1 symptoms, but these had a similar pattern as those of the highly susceptible cultivars expressing symptoms of yellowing. Not all test fields recorded similar levels of symptom expression. A cultivar sometimes had no symptoms in one test field, whereas grade 4 symptoms were recorded in several other test fields (e.g. cv. H 736110 at 300 d and 350 d). The median of the symptoms for all eight test fields calculated for each of the three cultivars at each inspection event showed three symptom peaks around days 200, 350 and 500, and possibly also a 4th peak at day 600 (Fig. 3). The frequency of symptom expression generally correlated with severity (Figs 2, 3). Because of the very different symptom severity in the different test fields, the quartile range of the data was rather wide. Therefore, a ‘symptom expression number’ was calculated in which symptom frequency and symptom grade were incorporated. For all 10 tested cultivars the symptom expression number agreed with the frequency of symptom expression (Fig. 4). The susceptible cultivars with high symptom scores (H65-7052, H 73-6110, H 87-4094) showed four peaks of symptom expression (days 200, 350, 500, and 600). The resistant or intermediately infected cultivars showed two or three symptom peaks, which occurred at the same plant age as the symptoms expressed by the susceptible cultivars. The strongest peak, which was around day 350, was visible in all cultivars (Fig. 4).

Correlation between presence of ScYLV and yellow leaf symptom expression Figure 2 Severity of yellow leaf symptom expression in sugarcane cultivars of different susceptibility to Sugarcane yellow leaf virusinfection. Symptoms of each variety in each of the eight test fields were rated on 14 occasions at approx. 40 d intervals. The number of symptom grades were added up for the whole growth period of ca. 650 d.

results also showed some cultivar-specific exceptions. The strongly infected cv. H 78-3606 exhibited few symptoms, as did the resistant cultivars, whereas the intermediately infected cv. H 65-7052 showed symptoms as severe as the strongly infected susceptible cvs H 73-6110 and H 87-4094.

Yellow leaf symptom expression during the growth period Yellow leaf symptoms were recorded in the eight test fields and are shown for three cultivars, two with high and one

A previous screening by tissue blot immunoassay classified the sugarcane cultivars according the presence of ScYLV (Table 1). This classification was confirmed in the present study where the presence of ScYLV was determined over the entire growth period (Fig. 4). All tested leaves of some cultivars tested positive for ScYLV over the whole life of the plant (H 73-6110, H 78-3567, H 87-4094), whereas others (H 78-4153, H 78-7750, H 82-3569, H 87-4319) rarely gave a positive reaction to ScYLV-antibody. Some cultivars gave variable reactions (H 65-7052, H 77-4643) while cv. H 78-3567 showed a continued low percentage of positive reactions. When the average ScYLV-infection rate and the average symptom expression were compared, a weak correlation (r2 = 0·50) was found (Fig. 5a). The correlation was weak because of two cultivars, H 65-7052, which had a low average ScYLV-infection but expressed symptoms, and cv. H 78-3606, with the opposite properties. In the two cultivars with variable immunoassay results (H 65-7052 and H 77-4643) occurrence of yellow leaf symptoms correlated with positive assays for presence of ScYLV Plant Pathology (2008) 57, 178–189

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Figure 3 Symptom expression of yellow leaf disease of three sugarcane cultivars in the eight test fields over the whole growth period. Average symptom grades of cultivars in the test fields represented by columns for each inspection event (MW = Maunawili, fd200 = field 200, fd604 = field 604, fd807 = field 807, fd330 = field 330, PK10 = Punakaawe 10, MM10 = Moomoku 10). No column of a particular field indicates no symptoms. The median (with upper and lower quartile range) for all fields is shown in the graphs on the right.

infection (Fig. 4). Cultivar H 65-7052 had infection peaks around (or slightly before) days 200, 300, 500 and 600, coincident with the four symptom peaks. Cultivar H 77-4643 had infection peaks at 150 d and 600 d, partly coincident with its symptom appearance at 200 d and 600 d after planting. A comparison between the positive ScYLV-infection and the symptom expression for all cultivars combined gave a better correlation (r2 = 0·67, Fig. 5b). A close correlation between ScYLV and yellow leaf symptoms was seen when virus-free and infected plants of Plant Pathology (2008) 57, 178–189

cv. H 87-4094, were planted side-by-side in the fields and compared. Symptom expression was 4-fold higher in the ScYLV-infected plants than in the virus-free plants, with yellow leaf-symptoms in 12·2% of stalks (88 out of 724) of infected plants, but only 3·2% of stalks (23 out of 722) of virus-free plants. In addition, none of the virus-free plants had symptoms exceeding grade 1. Shortly before harvest the plants from virus-free seed cane were tested again for ScYLV and 18% of the formerly virus-free plants became infected during their growth in the field (Lehrer et al., 2007).

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Figure 4 Presence of Sugarcane yellow leaf virus, symptom frequency and symptom expression over the growth period of all tested sugarcane cultivars. Susceptible cvs (H 73-6110, H 78-3606, H 87-4094), cvs with intermediately infected (H 65-7052, H 77-4643, H 78-3567) and resistant cvs (H 78-4153, H 78-7750, H 82-3569, H 87-4319) were inspected and tested for ScYLV at approx. 40 d intervals. The data from all eight fields were added at each inspection event. The symptom frequency indicates how often symptoms were observed irrespective of their grade. The symptom expression graph shows the average symptom grades per field.

Plant Pathology (2008) 57, 178–189

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Figure 5 Correlation between presence of Sugarcane yellow leaf virus and symptom expression over the whole growth period (a) and at the symptom expression peak phase (b). The infection status of the number 1 leaf and the symptom expression of the plants were determined at 40 d intervals. The average percentage of positively reacting blots per sampling event was calculated for each cultivar. The symptom expression was graded, grades were added up and the average was calculated. The data from all test fields were collected. (Correlation for (a) r 2 = 0·50 and for (b) r 2 = 0·67).

Comparison of fields for symptom expression One of the cultivars expressed severe symptoms in some of the test fields (Fig. 3). Therefore the test fields in the three Hawaiian islands were compared to see whether there is a ‘field factor’ (location, climate and soil) which influenced yellow leaf symptom expression (Table 2). Three field groups could be identified with respect to symptom expression. Field Moomoku 10, field 807 and field 604 showed relatively high symptom incidence (0·37–0·40), Punakaawe 10, field 330, field 200 and field Kunia had intermediate symptom incidence (0·21–0·27), and one field (Maunawili) had low symptom incidence (0·12). Similar trends were seen when the symptom grades were plotted for the different fields (Fig. 6). The low incidence field had lower grade symptoms and one of the high incidence fields also showed high symptom grades. No island-specificity was observed. Plant Pathology (2008) 57, 178–189

Figure 6 Severity of yellow leaf symptom expression in sugarcane cultivars in different test fields. The symptoms in each of the eight test fields were inspected and rated and the numbers of symptom grades of all cultivars in each field were added up for the whole growth period of ca. 650 d.

Coincidence of yellow leaf symptom expression and temperature The fluctuations of symptom expression in all cultivars, variations in ScYLV-presence in the two intermediately infected cultivars, and the correlation of test fields with symptom expression pointed to a possible climatic factor as elicitor. Seasonal fluctuations in symptoms were investigated (Figs 3, 4), although in Hawaii the seasonal differences are rather small. Seed pieces were planted in Maui and Oahu at the end of January, and in Kauai in mid June. If climate was mainly responsible for the symptom

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Symptom expression number

Test field

Field properties

Kauai Moomoku 10

150 m elevation, south-western mountain range, climate hot and cloudy, soil Makaweli silty clay loam, compact, water-logged 30 m elevation, south-western coastal plain, climate hot and dry, soil Makaweli silty clay loam and stony clay loam. 100 m elevation, south-western mountain range, climate extremely dry, windy, soil Makaweli silty clay loam, very fine. 50 m elevation, central plain climate very hot, windy, soil fine sand, very dry. 30 m elevation, lowland plain at north coast climate warm and windy, soil Ewa silty clay loam, fine. 200 m elevation, upper range of western mountain slope, climate warm and windy, soil Haliimale silty clay, fine, partly stony. 50 m elevation, leeward side, south part of central plain, climate very hot and dry, soil fine clay. 200 m elevation, windward side north-east mountain range, climate cool, windy, rainy, soil humous and clay.

Kauai Punakaawe 10 Kauai field 330 Maui field 807 Maui field 604 Maui field 200 Oahu Kunia Oahu Maunawili

Table 2 Symptom expression numbera of yellow leaf disease in different test fields in Hawaii

0·40

0·21

0·24

0·40

0·36

0·27

0·27

0·12

a

The symptom expression number was obtained by adding up all observed symptom grades in a test field and dividing the sum by the number of cultivars in the field and the number of inspection events.

outbreaks, then the dates of the symptom peaks in plants from Maui and Oahu should coincide with the dates of the symptom peaks in Kauai plants. However that correlation proved to be weak. The average difference in degree of symptom expression between Maui and Oahu plants and Kauai plants was 3·3 ± 1·2 when the symptoms were ordered according to date, and 1·6 ± 1·3 when ordered according to plant age. Therefore the correlation to plant age was better than to season. A simultaneous monitoring of air temperature and symptom occurrence in field 604 in Maui also gave no clear correlation (Fig. 7). The first peak of symptoms was clearly after the temperature maximum and the second symptom peak was after the temperature minimum.

Coincidence of yellow leaf symptom expression and nutrient or water shortage Because the test fields themselves exerted some influence on symptom intensity, some nutritional stress factors were tested for promotion of symptom expression. Plants of three highly susceptible cultivars (H 65-7052, H 73-6110, H 87-4094) were grown for 6 months in hydroponic sand culture. Some plants were subjected to mild drought by receiving only half of the daily water compared to control plants, others were fed with sodium chloride instead of calcium chloride, and others had ammonium sulphate in

the nutrient solution instead of ammonium phosphate. The plants with a shortage of calcium showed neither growth defects nor yellow leaf symptoms, except for cv. H 65-7052, which developed leaf yellowing after 5 months. However, symptoms also appeared in the control group of this cultivar. The plants under phosphate-shortage grew more slowly than the control plants and the growth of secondary shoots (tillers) was completely suppressed. Also in this test group, cv. H 65-7052 developed yellow leaf symptoms after 5 months both in the phosphate-deficiency and in the control group. The plants under water-shortage had reduced growth and showed drought symptoms such as dry leaf-ends. The yellow leaf symptoms appeared after 5 months in cv. H 65-7052, again under drought and under control conditions. Thus no nutritional factor was identified which gave rise to higher symptom incidence than in the control plants. Colonization of 3-month old virus-free cv. H 65-7052 by aphids, either virus-free M. sacchari and S. flava or viruliferous M. sacchari, did not elicit more or earlier yellow leaf symptoms in the following 2 months than aphid-free plants.

Discussion A clear relationship between the presence of ScYLV in sugarcane leaves and yellow leaf symptoms of the plant is Plant Pathology (2008) 57, 178–189

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Figure 7 Expression and severity of yellow leaf disease in three Sugarcane yellow leaf virussusceptible cultivars over the growth period and the prevalent ambient air temperatures. Cultivars H 65-7052, H 73-6110 and H 87-4094 were grown in a test plot in a commercial field on Maui. The symptom expression in the test plots and the air temperatures were recorded simultaneously.

demonstrated, as: (i) only susceptible and intermediately susceptible infected cultivars showed severe symptoms (grade 3 and above); (ii) the virus-free, resistant cultivars rarely showed yellow leaf symptoms and if they did, the symptoms were mostly of grade 1; (iii) a greater percentage of the ScYLV positive leaves of the intermediately infected cvs H 65-7052 and H 78-3567 coincided with the development of yellow leaf symptoms; and (iv) the ScYLVinfected plants of a susceptible cultivar showed a four times greater yellow leaf symptom expression rate than the virus-free plants of the same cultivar. The results confirm similar studies by Rassaby et al. (2003), who also found more frequent and stronger symptom expression in ScYLV-infected cane, especially after the first ratoon cycle. However, there was not a strict correlation between ScYLV and yellow leaf symptoms. For example, one of the cultivars with high ScYLV-infection expressed only weak yellow leaf symptoms (H 77-3606) and one cultivar with among the strongest symptoms had only an intermediate infection rate (H 65-7052). Furthermore, some cultivars consistently expressed a high percentage of infected leaves but the expression of symptoms fluctuated with plant age. Several aspects of these complications should be considered, e.g. how reliable was the ScYLV detection method, how reliable was the symptom determination and which factors besides ScYLV may have additionally evoked or suppressed symptom expression. The tissue blot immunoassay for ScYLV indicates the presence or absence of the virus, it does not determine the virus titre. However, differences in colour development in the standardized test were observed (data not shown). A threshold value of virus concentration for the positive reaction cannot be given, but it is thought to be very low because the virus is confined to the phloem of the leaf. Indeed, a strong TBIA reaction was observed where the ELISA assay of the leaf extract did not lead to a colour development with the same antibody. In the case of intermediately infected cultivars, the virus titre appeared to fluctuate above and below the detection threshold of the Plant Pathology (2008) 57, 178–189

test but the reason is not known. A variable percentage of ScYLV-infection in some cultivars tested by RT-PCR has also been reported for sugarcane cultivars in Réunion Island (Rassaby et al., 2004). RT-PCR is a more sensitive method than TBIA (Gonçalves et al., 2002; Korimbocus et al., 2002) and a very low presence of ScYLV has been recorded in even the so-called resistant varieties, but this does not invalidate the conclusions of this study. If virus infection is the cause of yellow leaf disease then the amount of ScYLV present in the plant matters, not whether traces of the virus may be found. Genotypic analysis has so far identified four ScYLV-genotypes which sometimes coexisted in the same sugarcane growing areas or even in the same cultivar (Moonan & Mirkov, 2002; Abu Ahmad et al., 2006a,b, 2007a,b). It is unknown whether these genotypes are equal in their ability to elicit symptom severity. Hawaiian cane was not included in that study. Aljanabi et al. (2001) concluded that marginal symptoms are not a reliable diagnostic indicator of ScYLV-infection since grade 1 symptoms, which may be elicited by (and confused with) non-pathogenic factors such as wounding or wind-damage, may potentially falsify results. In the present study, the carefully executed grading, rated the symptoms from grade 1 to grade 6 and the conclusions rely on the symptom grades 2–5. The fact that yellow leaf disease is observed as a sporadic event makes the elucidation of a strict correlation between symptoms and ScYLV as a single factor unlikely. But this is not a unique case for either a viral pathogen or other sugarcane pathogens. Banana streak badnavirus shows a loose correlation of symptom expression and infection, also depending on environmental factors such as temperature (Dahal et al., 1998, 2000). The same is reported with Sugarcane bacilliform virus in an Indian sugarcane collection (Viswanathan et al., 1996) and with sugarcane phytoplasmas in Australian sugarcane (Tran-Nguyen et al., 2000). ScYLV-infection together with plant and cultivation factors may have led to yellow leaf disease outbreak on Hawaiian fields. In the present study some

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field crops appeared to have frequent, severe symptom expression, while others had less. One of the severely affected fields had the highest drought stress due to hot climate, sandy soil and strong winds, whereas the crop with the lowest drought stress and the richest (humous) soil had low symptom expression. Overall symptom outbreak could not be correlated either to ambient air temperature or nutrient shortage. The involvement of phytoplasmas (which were not thoroughly investigated for Hawaiian cane) or the simultaneous presence of virus and phytoplasma cannot be ruled out at the moment (Cronje & Bailey, 1999; Aljanabi et al., 2001). Occurrence of yellow leaf symptoms showed four peaks in the infected cultivars, at 200, 350, 500 and 600 days after planting. One reason for the symptom fluctuations may lie in the yearly seasons although seasonal differences in Hawaii are small. The peaks might suggest a biannual ‘season’ for symptoms, for example, a summer drought and a winter cooling but test plants in Kauai, planted in June, and of test plants in Maui, planted at the end of January, did not coincide in the same months, which does not suggest a seasonal cause. Similarly, ambient temperature had no correlation with symptom outbreak. Alternatively peaks in symptom expression may be associated with plant-specific development stages and related physiological events. There are indications that ScYLVinfection slows the export of assimilates from the source leaves to the sink resulting in assimilates backing up in the source leaves, eventually inducing degradation of chlorophyll and chloroplasts resulting in leaf yellowing (Lehrer et al., 2001). Young plants may suffer less because the short distance between source and sink results in a steep assimilate gradient. In long stalks the assimilates may accumulate due to the lower pressure gradient in the phloem. When large primary sugarcane stalks lodge and side shoots (tillers and suckers) germinate, the new shoot formation, with its strong sink activity and fast growth, may alleviate the back-up of assimilates in the source leaves and prevent further leaf yellowing. When the side shoots have grown taller their source leaves may again suffer the same assimilate export limitation as the primary stalk did before, and the next phase of leaf yellowing may start. The gap of about 150 d between the symptom peaks corresponds to the time needed to grow a top of totally new green leaves (10 d intervals per leaf and about 13– 15 leaves per leaf-top). This assimilate backup in lodging sugarcane may be further enhanced by lower temperature. It was found previously that cooling has a strong effect on long distance transport of assimilates, stronger than on photosynthesis or sugar synthesis, with the result that the sugar content in the leaves increased at low temperature (Ebrahim et al., 1998). Although the correlation of symptom outbreak with infection status is not consistent, the results presented here indicate that ScYLV-infection is most likely the cause of symptoms of yellow leaf disease. Further studies are being directed to leaf yellowing in infected sugarcane to reveal the interference of ScYLV on the metabolism of phloem cells.

Acknowledgements The authors are grateful for the help provided by field workers of Hawaiian Commercial and Sugar (HC&S, Maui), workers of Gay & Robinson (Kauai), and workers of Hawaii Agriculture Research Center (HARC, Aiea). The work was funded by Deutsche Forschungsgemeinschaft, Hawaii Agriculture Research Center, HC&S and Gay & Robinson. The authors gratefully acknowledge the gift of antibody against ScYLV from BEL Lockhart and correction of the manuscript by Dr S Schenck, HARC.

References Abu Ahmad Y, Rassaby L, Royer M et al., 2006a. Yellow leaf of sugarcane is caused by at least three different genotypes of Sugarcane yellow leaf virus, one of which predominates on the Island of Reunion. Archives of Virology 151, 1355 –71. Abu Ahmad Y, Royer M, Daugrois J-H et al., 2006b. Geographical distribution of four Sugarcane yellow leaf virus genotypes. Plant Disease 90, 1156 –60. Abu Ahmad Y, Costet L, Daugrois J-H et al., 2007a. Variation in infection capacity and in virulence exists between genotypes of Sugarcane yellow leaf virus. Plant Disease 90, 253 – 9. Abu Ahmad Y, Girard J.-C, Fernandez E et al., 2007b. Variation in virus populations and growth characteristics of two sugarcane cultivars naturally infected by Sugarcane yellow leaf virus in different geographical locations. Plant Pathology 56, 743–54. Aljanabi SM, Parmessur Y, Moutia Y, Saumtally S, Dookun A, 2001. Further evidence of the association of a phytoplasma and a virus with yellow leaf syndrome in sugarcane. Plant Pathology 50, 628 –36. Bailey RA, Bechet GR, Cronje CPR, 1996. Notes on the occurrence of yellow leaf syndrome of sugarcane in southern Africa. South African Sugar Technology Association Proceedings 70, 3 – 6. Chatenet M, Delage C, Ripolles M, Irey M, Lockhart BEL, Rott P, 2001. Detection of Sugarcane yellow leaf virus in quarantine and production of virus-free sugarcane by apical meristem culture. Plant Disease 85, 1177 –80. Comstock JC, Irvine JE, Miller JD, 1994. Yellow leaf syndrome appears on the United States mainland. Sugar Journal 56, 33 –35. Comstock JC, Irey MS, Lockhart BEL, Wang ZK, 1998. Incidence of yellow leaf syndrome in CP cultivars based on polymerase chain reaction and serological techniques. Sugar Cane 4, 21 –4. Cronje CPR, Bailey RA, 1999. Association of phytoplasmas with yellow leaf syndrome of sugarcane. In: Proceedings of the 23rd Congress of the International Society of Sugarcane Technologists, New Delhi, India, 1999, 373 –81. Dahal G, Hughes Jd′A, Thottappilly G, Lockhart BEL, 1998. Effect of temperature on symptom expression and reliability of banana streak badnavirus detection in naturally infected plantain and banana (Musa spp.). Plant Disease 82, 16 –21. Dahal G, Ortiz R, Tenkouano A, Hughes Jd′A et al., 2000. Relationship between natural occurrence of banana streak badnavirus and symptom expression, relative concentration

Plant Pathology (2008) 57, 178–189

Sugarcane yellow leaf and ScYLV infection

of viral antigen, and yield characteristics of some micropropagated Musa spp. Plant Pathology 49, 68– 79. Ebrahim MK, Zingsheim O, El-Shourbagy MN, Moore PH, Komor E, 1998. Growth and sugar storage in sugarcane grown at temperatures below and above optimum. Journal of Plant Physiology 153, 593–602. Correction 154, 416. Fitch MMM, Lehrer AT, Komor E, Moore PH, 2001. Elimination of Sugarcane yellow leaf virus from infected sugarcane plants by meristem tip culture visualized by tissue blot immunoassay. Plant Pathology 50, 676– 80. Gonçalves MC, Klerks MM, Verbeek M, Vega J, van den Heuvel JFJM, 2002. The use of molecular beacons combined with NASBA for the sensitive detection of Sugarcane yellow leaf virus. European Journal of Plant Pathology 108, 401– 7. Korimbocus J, Coates D, Barker I, Boonham N, 2002. Improved detection of Sugarcane yellow leaf virus using a real-time fluorescent (TaqMan) RT-PCR assay. Journal of Virological Methods 103,109– 20. Lehrer A, Meinzer R, Moore P, Komor E, 2001. Physiological consequences of Sugarcane yellow leaf virus infection on the sugarcane plant. In: Hogarth DM, ed. Proceedings of the XXIV Congress of the International Society of Sugar Cane Technologists, Vol II. ASSCT Mackay, Australia: Australian Society of Sugar Cane Technologists, 657– 9. Lehrer AT, Schenck S, Yan S-L, Komor E, 2007. Movement of aphid-transmitted Sugarcane yellow leaf virus (ScYLV) within and between sugarcane plants. Plant Pathology 56, 711–7. Martin JP, Eckert RC, 1925. Macronutrients and trace elements required for sugar cane green house cultivation. Hawaiian Planters Record 29, 424– 49. Matsuoka S, Meneghin SP, 1999. Yellow leaf syndrome and alleged pathogens: a casual but not a causal relationship. In: Proceedings of the 23rd Congress of the International Society of Sugar Cane Technologists New Delhi, India, 1999, 382 –9. Moonan F, Mirkov TE, 2002. Analyses of genotypic diversity among North, South, and Central American isolates of Sugarcane yellow leaf virus: evidence for Colombian origins

Plant Pathology (2008) 57, 178–189

189

and for intraspecific spatial phylogenetic variation. Journal of Virology 76, 1339 –48. Parmessur Y, Aljanabi S, Saumtally S, Dookun-Saumtally A, 2002. Sugarcane yellow leaf virus and sugarcane yellows phytoplasma: elimination by tissue culture. Plant Pathology 51, 561 –6. Rassaby L, Girard J-C, Lemaire O et al., 2004. Spread of Sugarcane yellow leaf virus in sugarcane plants and fields on the island of Réunion. Plant Pathology 53, 117 –25. Rassaby L, Girard J-C, Letourmy P et al., 2003. Impact of Sugarcane yellow leaf virus on sugarcane yield and juice quality in Réunion Island. European Journal of Plant Pathology 109, 459 –66. Scagliusi SM, Lockhart BEL, 2000. Transmission, characterization, and serology of a luteovirus associated with yellow leaf syndrome of sugarcane. Phytopathology 90, 120 – 4. Schenck S, 1990. Yellow leaf syndrome – a new sugarcane disease. Hawaiian Sugar Planters Association: Annual Report. Schenck S, Lehrer AT, 2000. Factors affecting the transmission and spread of Sugarcane yellow leaf virus. Plant Disease 84, 1085 –8. Schenck S, Hu JS, Lockhart BEL, 1997. Use of a tissue blot immunoassay to determine the distribution of sugarcane yellow leaf virus in Hawaii. Sugar Cane 4, 5 – 8. Sholto Douglas JWEH, 1956. The application of hydroponics to the sugar industry. The Sugar Journal 19, 30–1. Tran-Nguyen L, Blanche KR, Egan B, Gibb KS, 2000. Diversity of phytoplasmas in northern Australian sugarcane and other grasses. Plant Pathology 49, 666 –9. Vega J, Scagliusi SMM, Ulian EC, 1997. Sugarcane yellow leaf disease in Brazil: evidence of association with a luteovirus. Plant Disease 81, 21 –6. Viswanathan R, Alexander KC, Garg ID, 1996. Detection of sugarcane bacilliform virus in sugarcane germplasm. Acta Virologica 40, 5 – 8.