Abstract: Sorghum ergot, caused by Claviceps africana Frederickson, Mantle and de Milliano, is a disease that poses a serious threat to sorghum, especially in hybrid seed production. The initial sign of the disease is called sphacelium that contains macroconidia that could play a role in the survival of the pathogen. Sorghum A-line ATx623 was planted in the greenhouse during 2001, 2002 and 2003 at College Station, Texas. Flowering panicles were inoculated until runoff with a suspension of 1.6x106 C. africana conidia mL-1. Sphacelia were collected at several stages depending on their maturity. Petri dish plates containing sphacelia were arranged in a factorial experiment with 16 treatments out of the combination of sphacelia maturity and temperature. Every month a conidia germination test was made. Conidia located on the sphacelium surface had greater germination than the conidia located inside the sphacelium. This may be due to the developmental maturity of the conidia located on the outside. Warmer storage temperatures (21°C) significantly reduced conidia viability compared with freezing or cool temperatures. Dry and cool temperatures are required to preserve conidia viability and newly-formed sphacelia have the highest conidial viability especially if conidia are located on the sphacelium surface. However, they show a greater viability reduction through time compared with conidia from older sphacelia, showing that conidial maturity can play a role on the survival of the conidia.
INTRODUCTION
Losses due to sorghum ergot caused by Claviceps africana Frederickson, Mantle and de Milliano in seed production fields can be high. In India, losses up to 80% have been reported in seed production fields whereas in Zimbabwe the annual losses are between 12 and 25% and sometimes up to 100% (Bandyopadhyay et al., 1998). In 1997, nearly 45% of the hybrid seed production fields in the Texas Panhandle had ergot with varying degrees of severity (Workneh and Rush, 2003). Losses from import rejection can occur. For example, in 1999, the Nicaraguan Inspection and Certification Department intercepted seeds with honeydew and sphacelial tissues mixed with seed in a shipment from the USA. This shipment was quarantined, resulting in losses of millions of dollars to seed companies.
Usually, this disease is not important in hybrid grain sorghum fields. Losses of seed quality can be an issue, because of honeydew contamination of healthy sorghum grain, increasing colonization by saprophytic fungi. McLaren (1992) found such seed had reduced germination. In addition, honeydew stickiness can interfere with harvest.
Many pathogenic ascomycetes that produce resting structures generate ascospores following carpogenic germination. There are differences among the maturity of such structures and their capability to survive. Within a crop, C. africana produces sphacelia, or perhaps also sclerotia. At harvest, sphacelia differ in age or have different degrees of sclerotial tissue development. Survival of the pathogen may be affected by the level of fungal development or by environmental conditions. Bhuiyan et al. (2002b) showed that C. africana macroconidia present in sorghum panicles that were held above soil surface survived for more than eight months over winter, suggesting that local survival can provide inoculum for future epidemics in Australia. Storage of sphacelia at high temperature (>32°C) resulted in a rapid decrease in viability of C. africana macroconidia with no spores viable after two weeks of storage. Conidia germinated after 17 weeks storage at 20°C. The effect of cool temperatures (6°C) were evaluated by Odvody et al. (1999), who observed that conidia of newly-formed sphacelia maintained viability at its maximum up to 12 weeks and then decreased 50% at 22 weeks of storage. In other study, Prom et al. (2005) showed that conidia located on sphacelia that were held above soil surface for a year survived and infected sorghum florets on male-sterile line ATx623.
The problem of honeydew on the surface of sorghum seeds had been addressed by Dahlberg et al. (1999), who found that contact fungicides captan (Captan 400®) and thiram (42-S Thiram®) were effective in inhibiting conidiophore and secondary conidia formation without drastically reducing the viability of the sorghum seed (1-4%). Frederickson and Odvody (2003) observed that conidia viability of newly intact sphacelia treated with captan (Captan 400®) was significantly reduced (63%) and cores from treated sphacelia did not show a major reduction compared with the control. They suggested that this could be due to the slight penetration ability of captan within the sphacelia or desiccation of the sphacelia.
In the sorghum hybrid seed production industry, the main goal is to obtain and maintain sorghum seed with high viability. Nevertheless, viability is highly influenced by storage conditions. The most critical conditions for seed in storage are low seed moisture and low temperature. In addition to these factors, seeds need to be free from inert material such as plant debris that could contain pathogens and insects.
Pathogens that are carried in seed lots may be either internally seed-borne or present on the seed surface, in plant debris, or infected weed seeds. These pathogens can survive long periods of storage along with dried seeds in a dormant stage and usually do not resume activity until seeds germinate (Gerard, 1984; Gilbert et al., 1997). Bhuiyan et al. (2002b) demonstrated that macroconidia of C. africana can survive on honeydew-coated seed for more than 12 months at 4°C (42-100% RH), suggesting that international seed exchange was a possible route for the accidental introduction of this pathogen to Australia (Komolong et al., 2002). Since ergot was detected during 1997 in the Texas seed production area, it is possible that pathogen structures could be spread with shipments exported overseas, possibly to sorghum producing areas that are free of the pathogen. Ellis (1984) reports that temperature has a dramatic effect on seed longevity and concluded that each 5°C reduction in seed temperature doubles the life of seeds. However, low temperatures can maintain pathogen viability. The objectives of this study were to determine the effect of storage temperature on the viability of C. africana macroconidia located on the surface and within the sphacelium and to observe the effect of sphacelium age on the macroconidial survival.
MATERIALS AND METHODS
Sorghum A-line ATx623 was planted in the greenhouse during 2001, 2002 and 2003 at College Station, Texas. Conidia were collected from a local C. africana isolate that was fresh maintained under greenhouse conditions in College Station. The greenhouse conditions to increase the inoculum were above 80% relative humidity and 30°C. Flowering panicles of ATx623 were tagged and inoculated with the local isolate by hand atomizer until runoff with the suspension of 1.6x106 C. africana conidia mL-1. Several panicles were selected according to their sphacelial development 7 to 10 days after inoculation. The greenhouse conditions during the development of the sphacelia were 50% relative humidity and 30°C. Sphacelia were collected at several stages depending on their maturity. Sphacelial structures were grouped into four maturity classes based on sphacelium development:
Class 1: Newly-formed sphacelia showing slightly transparent honeydew ooze.
Class 2: One-week-old sphacelia showing high quantities of transparent honeydew ooze.
Class 3: Two-week-old sphacelia with dark-brown dried honeydew.
Class 4: Three-week-old sphacelia showing hardness on the sphacelia surface and honeydew crust.
Sphacelia, attached to the panicle rachis were placed in petri dishes containing color silica gel (as a dessicant). Every time that the silica gel showed changes in color, it was changed for a new one (this was done to ensure that the relative humidity inside the dish plate was low). Dishes were sealed with parafilm. The incubation temperatures of sphacelia were fluctuating sub-freezing (0 to -3°C), 7, 14 and 21°C. Dishes were arranged in a factorial experiment with 16 treatments out of the combination of sphacelia maturity (four levels) and temperature (four levels); each plot was replicated four times in a randomized complete design. The model used was:
Yijk = μ + αi + βj
+ αβij + εijk |
Where μ is the overall mean conidia germination, αi is the effect of the ith level of temperature, βj is the effect of the jth level of sphacelia age and αβij is the interaction effect of the ith level of temperature with the jth level of sphacelia age. Conidia survival was measured as the proportion of germinated macroconidia showing conidiophore formation with secondary conidia at their tips and was measured almost every month by sampling macroconidia located on the sphacelium surface and within the sphacelium interior. A random sample of 20 sphacelia was taken from each one of the treatments and after removing plant tissue, was placed into vials containing 20 mL distilled water. Vials were stirred for one minute and a portion of the suspension (1 mL) was placed onto water-agar plates. Four replications per treatment were made and incubated overnight at room temperature (21°C). The germination observed in this sample was named germination on the sphacelium surface. After rinsing the remaining sphacelia with a jet of water for 30 sec, they were macerated using a mortar, suspended in 10 mL distilled water, stirred for 30 sec and placed onto water-agar plates. The germination percentage obtained here was named within the sphacelium. Original data was transformed using the arcsine of the square root of each value to comply with normality distribution assumptions. To determine significant differences between means, Tukey's mean separation was used at p<0.01. A Chi square test was performed to see if the variances between years were homogeneous.
RESULTS
Out of the three years of study, two variances were homogeneous. By this result the data for the three year study was combined. There was a highly significant effect of the main factor temperature across the six month study on germination of Claviceps africana conidia located on the sphacelium surface, while sphacelium age had a highly significant effect up to the fifth month, same as the interaction between these two factors (Table 1). Almost the same results were obtained in the ANOVA table for germination of conidia located within the sphacelium, that was affected by both factors up to the fifth month (Table 2). Warmer storage temperatures (21°C) significantly reduced germination across the 6-month period in conidia located on the sphacelium surface. The reduction ranged from 42 to 100% compared with frozen temperatures and from 26 to 100% compared with cool temperatures (7°C). At the end of the 6-month study, frozen temperatures show the highest significant conidia germination, with a reduction of 59% in that period. Conidia on younger sphacelium showed significantly more germination (47-65%) compared with older sphacelium conidia in the first 3-month period. However, this situation was reversed in the second 3-month period where newly-formed sphacelium gave the lowest conidia germination. At the end of the 6-month trial, all sphacelial ages gave statistically the same conidia germination on the sphacelium surface (Table 3). Identical situations were observed in conidia located within the sphacelium, where the warmer treatment reduced conidia viability from 46 to 100% compared with frozen temperatures and newly formed sphacelia showed 42 to 73% more conidia germination than older sphacelia in the first 3-month period. After that, conidia germination was statistically similar in both sphacelial ages (Table 4).
Comparing the average germination across years and dates in both conidia locations,
conidia from the sphacelium surface had more germination at all levels of storage
temperature and sphacelium age (Table 5) than interior conidia.
| Table 1: | Observed mean squares and test of significance of main factors on germination of C. africana conidia located on the sphacelium surface |
| ** = Highly significant effect at p<0.01; * = Significant effect at p<0.05; ns = Not significant | |
| Table 2: | Observed mean squares and test of significance of main factors on germination of C. africana conidia located within the sphacelium |
| ** = Highly significant effect at p<0.01; ns = Not significant | |
| Table 3: | Effect of main factors on the average C. africana conidia germination located on the sphacelium surface |
| * = Treatments with the same letter(s) in each category are statistically similar according to Tukey (p<0.01) | |
| Table 4: | Effect of main factors on the average C. africana conidia germination (%) located within the sphacelium |
| * = Treatments with the same letter(s) in each category are statistically similar according to Tukey (p<0.01) | |
| Table 5: | Effect of storage temperature and sphacelium age on germination of C. africana conidia located on the surface and within the sphacelial tissue |
| * = Treatments with the same letter(s) in each category are statistically similar according to Tukey (p<0.01) | |
Conidia from within the sphacelium and the sphacelium surface stored at warmer
conditions showed a significant reduction of 75 and 73.5%, respectively compared
with frozen temperatures in the same location. Also, conidia from younger sphacelium
showed 50 to 53% more germination than conidia from older sphacelium in both
conidia locations. The combined analysis showed significant differences among
storage temperatures at each sphacelium age. At all sphacelia ages, conidial
germination decreased as temperature increased from 0 to 21°C (Table
6). This suggests that temperatures at locations with summer-fall planting
dates could promote survival of conidia outside and inside the sphacelia, creating
a viable source of inoculum for the next crop season.
| Table 6: | Effect of the interaction between sphacelium age and storage temperature on germination of C. africana conidia |
The sphacelial age effect on conidia germination showed that conidia from the surface of newly formed sphacelium had statistically more germination than the other sphacelia ages during the first three months and then decreased after this time. During the 6-month period, newly-formed sphacelia had half the conidial germination of the older sphacelia for every unit increase of time. Viability of conidia located within newly-formed sphacelia was higher during the first two months. Older sphacelia had lower viability of conidia up to the fourth month. Conidia on newly-formed sphacelia decline in viability three times faster than the oldest sphacelia. Cooler storage temperatures showed significantly highest viability values through the 6-month period with 42 to 99% higher than the warmest temperature. Conidial germination rate declined 5 times faster at the warmest storage temperature as compared with the coolest. Conidial viability at the 21°C treatment was nil at sixth month, while conidia viability was more than 10% at cooler storage after the sixth month. Similar trends were observed with conidia from within the sphacelia. However, the viability was lower at all the storage temperatures. The warmest storage temperature showed 4.5 times more reduction in the conidial germination as compared with the coolest and twice compared with the 14°C.
In general, the interaction showed that conidia from younger sphacelia maintained statistically a high viability if they were exposed to cool temperatures of 0 to 7°C (Table 6).
DISCUSSION
This study shows that survival of conidia of older sphacelia, which are most common during harvest of commercial or seed production fields, is very sensitive to the warmer temperatures that would be present during the summer in spring-planted sorghum production areas. Where cooler temperatures prevail following a crop (e.g., with summer or fall-planted crops), conidia may survive longer, perhaps contributing to local survival of inoculum for the next crop season. Conidia located on the sphacelium surface had greater germination than the conidia located inside the sphacelium. This may be due to the developmental maturity of the conidia located on the outside. Warmer storage temperatures (21°C) significantly reduced conidia viability compared with freezing or cool temperatures (<21°C). Dry and cool temperatures are required to preserve conidia viability and newly-formed sphacelia have the highest conidial viability especially if conidia are located on the sphacelium surface. Averaged over all sphacelia ages, conidial viability decreased as temperature increased from 0 to 21°C (r = -0.75 at p<0.0009). Similar results obtained by Odvody et al. (1999) showed that C. africana macroconidia maintained viability stored at 6°C, with a maximum up to 12 weeks and then decreased 50% at 22 weeks of storage, whereas Bhuiyan et al. (2002b) showed that storage of sphacelia at high temperature (>32°C) resulted in a rapid decrease in viability of C. africana macroconidia, with no spores viable after two weeks of storage.
Conidia from the surface of newly-formed sphacelia had statistically more germination than the other sphacelial ages during the first three months. During the 6-month period, newly-formed sphacelia had a reduction in conidial viability of twice the value of the older sphacelia for every increase unit of time. Conidia viability at the 21°C treatment declined to zero at the sixth month, while conidial viability at cooler temperatures were more than 10% at the sixth month. Similar trends were observed with the conidia from within the sphacelia. Conidial viability at the highest storage temperature was eliminated at the sixth month. These results are similar to those of Bhuiyan et al. (2002a), who found that C. africana conidia showed little germination after 17 weeks storage at 20°C. Also, conidia survived for more than eight months stored outside over the winter months. These results support those of Prom et al. (2005), who showed that conidia could be viable up to 12 months under field conditions in Texas. Therefore, we can conclude that environmental conditions affecting viability of sphacelia stored under cool temperatures maintained conidial viability and newly-formed sphacelia located on the sphacelia surface had the highest conidial viability. However, they show a greater viability reduction through time compared with conidia from older sphacelia, showing that conidial maturity can play a role on the survival of the conidia and perhaps in the new infections that will develop on the following crop season.