The conservation of Ustilago tritici in infected seed

Abstract

Traditional methods of maintaining strains of Ustilago tritici over long periods of time consume large amounts of labour and physical resources, with variable results. These methods generally involve the storage of teliospores of each isolate at temperatures of 2–4 ^oC. Very little long-term work has been conducted on the survival of mycelium of U. tritici in infected host seed. Our objective was to determine the survival of race T2 of U. tritici as mycelium in infected wheat seed stored at –15 ^oC for up to 32 years. There was a significant decline in the per cent viability of the inoculated wheat seed over time. Conversely, there was a significant increase in the per cent infection of the viable seed over time. This suggests that either the cold treatment enhances the survival of the mycelium of the pathogen, or the fungus enhances the survival of the wheat seed at low temperatures. Storage of U. tritici as mycelium in infected seed at –15 ^oC is an effective technique for long-term preservation of this pathogen.

Résumé: Les méthodes traditionnelles permettant de conserver les souches d'Ustilago tritici pendant de longues périodes exigent beaucoup de travail et de ressources et, malgré tout, les résultats obtenus sont mitigés. Ces méthodes impliquent habituellement l'entreposage des téleutospores de chaque isolat à une température variant de 2 à 4 ˚C. Peu de travaux ont été faits sur la survie du mycélium d'U. tritici dans les semences hôtes infectées. Notre objectif était d'évaluer la survie de la race T2 d'U. tritici sous forme de mycélium dans des semences de blé entreposées à –15 ˚C pour au moins 32 ans. [Agrave] la longue, il y a eu un déclin notable du taux de viabilité des semences de blé. Par ailleurs, il y a eu une augmentation marquée du taux d'infection chez les semences viables. Cela suggère soit que le traitement par le froid prolonge la survie du mycélium de l'agent pathogène, soit que le champignon prolonge la survie des semences de blé entreposées à basse température. L'entreposage d'U. tritici à –15 ˚C, sous forme de mycélium dans des semences infectées, est un moyen efficace de préserver cet agent pathogène sur une longue période.

Keywords:

• cold storage
• inoculum survival
• smut
• wheat

Mots clés:

• blé
• charbon
• entreposage sous froid
• survie de l'inoculum

Introduction

The collection of isolates of Ustilago tritici (Pers.) Rostr. from western Canada is normally conducted during annual surveys of farmers' fields (Menzies et al., 2007). Individual smutted heads are collected from each field with smutted plants, for eventual determination of the race composition of the pathogen population (Nielsen, 1987; Menzies et al., 2003). This work was initiated in the early 1930s (Hanna, 1937), and has been conducted on a regular basis since 1964 (Menzies et al., 2003). The information obtained is invaluable for determining which host resistance genes are necessary for incorporation into new wheat lines and detecting how the virulence of the pathogen population has changed over time. The collection of the pathogen isolates has also been useful in genetic studies of the host and pathogen (Nielsen, 1988). The preservation of new and interesting genotypes of U. tritici from western Canada and other parts of the world can be a problem, however, because traditional methods are very laborious and time consuming.

Sori of different races of U. tritici on wheat spikes are normally dried and stored in glass jars at 2–4 ^oC at the Cereal Research Centre (CRC), Winnipeg, MB (Nielsen, 1987). Teliospores stored in this way can survive with acceptable germination rates (greater than 50%) for up to 16 years, but results are quite variable. Survival during storage in this manner for up to 10 years cannot be assured (Sampson, 1928; Nielsen, 1987; Menzies et al., 1997). Similarly, U. nuda smutted heads of barley stored in glassine bags or paper envelopes in a refrigerator can remain viable for 1–10 years (Tapke, 1955). Rejuvenation from a teliospore collection involves inoculation of host florets to obtain infected seed and the growing of the infected seed to flowering to obtain the new generation of teliospores. This process can take up to 200 days. Valuable greenhouse and growth cabinet space is occupied during this process. A technique for longer-term storage of U. tritici is desirable to ensure survival of important genotypes and reduce the resources needed to properly preserve different genotypes of the pathogen.

There have been reports in the literature of strains of seed-infecting Ustilago spp. surviving as mycelium in infected seed. Studies with U. nuda have shown that this fungus can survive in infected barley seed for 2–10 years of storage under various storage conditions (Tapke, 1955; Russell, 1961; Roszko et al., 1980). Tiemann (1925) showed that per cent infected plants decreased only slightly over three years from U. tritici infected wheat seed stored at room temperature. Buchheim (1935) found that storage of U. tritici-infected wheat seed below 0 ^oC did not affect the percentage of smutted plants arising from that seed. These findings, along with the predictions of Roberts & Ellis (1977) that wheat seed stored at –20  ^oC at a 5% moisture content would have only a 5% decrease in germination rate after 78 years, suggest that U. tritici mycelium in infected wheat seed may be able to survive for a long period of time.

The objective of these experiments was to determine the ability of U. tritici to survive in infected wheat seed for 30 years at –15 ^oC. A summary of the first 20 years of this study was previously reported (Menzies et al., 1997).

Materials and methods

The plant growth conditions and methods used for inoculation with U. tritici were previously reported by Menzies et al. (1997). Briefly, race T2 of U. tritici was inoculated onto spikelets of the wheat differential TD-3 using the needle inoculation method (Nielsen, 1987) in February 1976. The seed obtained from the inoculated spikelets were stored in a desiccator at room temperature for one week. Sixty seeds were removed and planted in soil beds in a greenhouse to determine the percentage of the viable seed (hereafter referred to as per cent viable seed) and the per cent of plants that displayed sori of U. tritici at plant maturity (hereafter referred to as per cent infected plants). The desiccator and the remaining seed were placed in a freezer at –15 ^oC for storage. Every two years, 60 seeds were removed from storage and planted in soil beds in a greenhouse to determine seed viability and the proportion of infected plants. The experiment ended in 2008, when the supply of inoculated seed was exhausted. The data were analyzed by regression analysis using SAS 9.1 Proc Reg (SAS Institute Inc, 2003), comparing the time of storage with seed viability and per cent infected plants.

Results and discussion

The results are presented in Table 1. The percentage of viable seed varied from 83% at eight years of storage to 35% after 28 years of storage. The low percentage viability of wheat seed in these experiments at time 0 may reflect seed dormancy issues, and we conclude that the original viability of the seed was approximately 85–90%. The viability at the end of the experiment was closer to 45–50%. There was a negative relationship (P < 0.0031, R² =  0.4523) between the time of storage and the percentage of viable seed. Roberts & Ellis (1977) predicted that wheat seed stored at –20 ^oC and 5% moisture content would survive long periods of time, with only a 5% drop in viability after 78 years. Our conditions are not quite those specified by Roberts & Ellis, but our wheat seed suffered a significant decline in viability after 32 years of storage. Nevertheless, this level of survival of the wheat seed would allow storage of mycelium of U. tritici in wheat seed for at least 30 years.

Table 1.  Percentage of plants produced and the percentage of these plants smutted from samples of 60 seeds of wheat differential TD-3 inoculated with race T2 of Ustilago tritici and stored at –15 ^oC since 1976

Number of years in storage	Per cent viable seed	Per cent infected plants
0	57	56
2	81	86
4	80	89
6	65	74
8	83	84
10	77	80
12	82	84
14	82	59
16	75	91
18	67	98
20	78	91
22	55	94
24	58	94
26	50	87
28	35	81
30	43	89
32	53	91

The percentage of plants with smutted heads varied from 56–98% and there was a positive relationship (P < 0.0486, R² = 0.2350) between the time of storage and the percentage of smutted plants (Fig. 1). This relationship was first observed after 20 years of storage (Menzies et al., 1997). The increase in the percentage of plants with smutted heads is encouraging for storing mycelium of U. tritici in infected seed, but the mechanism behind an enhanced survival of infected seed is unknown. It is possible that the survival of mycelium of the pathogen may be enhanced by the cold treatment, or the pathogen may positively influence the survival of the infected seed. Other studies have not reported this phenomenon, however. Tiemann (1925) observed that the levels of infection of wheat plants grown from U. tritici-infected seed stored at room temperature for three years decreased slightly, while Buchheim (1935) found that storage of infected wheat seed at 0 ^oC had no effect on the percentage of smutted heads. Tapke (1955) noted survival of U. nuda in infected barley seed stored at –2 to 0 ^oC for seven years, with a 73% level of smutted plants. However, he did not report the initial level of smutted plants from the infected seed. Russell (1961) saw a reduction in the level of smutted plants arising from some samples of U. nuda-infected barley seed stored in glass jars at room temperature for six years, while other samples did not show a decline after 10 years of storage. Russell attributed the decline in viability of U. nuda-infected seed in some samples to weaker embryos in the infected seed. Roszko et al. (1980), also working with U. nuda and barley, observed that U. nuda-infected seed did not survive storage well. This resulted in a decrease in the per cent of the viable seed producing plants with smutted heads over time. Roszko et al. (1980) recommended that U. nuda-infected seed not be stored any longer than two years to ensure survival of the pathogen. The reasons for differences among these studies are unclear. It should be noted that levels of smutted plants arising from infected seed can be extremely variable (Thomas, 1991). Some of the differences in levels of smutted plants referred to by the different authors may not be real differences. They may simply reflect the expected variability of the percentage of smutted plants inherent in these types of experiments. Our study was considerably longer than any of the other studies. Any long-term effects of storage of U. tritici-infected seed would become more manifest over the 32 years.

Fig. 1. The effect of storage of wheat seed infected with race T2 of Ustilago tritici at –15 ^oC on the subsequent percentage viability of the wheat seed and the percentage of plants with smutted heads arising from the germinating seed. There was a negative relationship (P < 0.0031, R² = 0.4523, y =  82.25  + –1.01x) between the time of storage and the percentage of viable seed and a positive relationship (P < 0.0486, R² = 0.2350, y  =  75.12 + 0.56x) between the time of storage and the per cent infected plants.

The survival of U. tritici in infected wheat seed for 32 years in our experiments indicates that this may be an efficient and effective technique for storing important isolates or genotypes of this pathogen. It is quite likely that the pathogen can survive significantly longer than 32 years in infected seed stored at –15 ^oC, but our seed supply was exhausted. All races of U. tritici and U. nuda are currently being stored in this manner at the CRC. The storage of these pathogens in infected seed at –15 ^oC consumes considerably less time and space than traditional storage techniques.

Notes

^†These authors are retired.