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2011. Plant Management Network. This article is in the public domain.
Accepted for publication 20 December 2010. Published 10 February 2011.


Mycobiota on Wild Oat (Avena fatua L.) Seed and Their Caryopsis Decay Potential


L. Z. de Luna, Pest Management Regulatory Agency, Health Canada, Ottawa, Ontario, Canada K1A 0K9; A. C. Kennedy and J. C. Hansen, Land Management and Water Conservation Research Unit, USDA-ARS, Pullman, WA 99164-6421; T. C. Paulitz, Root Disease and Biological Control Research Unit, USDA-ARS, Pullman, WA 99164-6421; R. S. Gallagher, Crop and Soil Sciences, Penn State, University Park, PA 16802-6211; and E. P. Fuerst, Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99164-6420


Corresponding author: A. C. Kennedy. akennedy@wsu.edu


de Luna, L. Z., Kennedy, A. C., Hansen, J. C., Paulitz, T. C., Gallagher, R. S., and Fuerst, E. P. 2011. Mycobiota on wild oat (Avena fatua L.) seed and their caryopsis decay potential. Online. Plant Health Progress doi:10.1094/PHP-2011-0210-01-RS.


Abstract

Wild oat is a serious weed in cereals that is difficult to control due to long-term survival in the weed seed bank. The mycobiota associated with dormant wild oat (Avena fatua L.) seeds buried for six months in a no-till wheat field were evaluated for their caryopsis decay potential. Of the 118 representative isolates tested, only 15% were found to have caryopsis decay potential. One isolate of Fusarium avenaceum and three isolates of Fusarium culmorum completely decayed wild oat caryopses within two weeks. Only a few isolates were susceptible to the antifungal activity from water or acetone extracts of wild oat hulls, suggesting that soluble chemicals from the hull play a minor role in resistance to decay. The procedures developed here can be used to isolate and screen individual organisms to determine their potential for seed decay and weed biocontrol.


Introduction

Wild oat (Avena fatua L.) is an annual grass weed that is particularly troublesome in cereal crops throughout North America, Europe, and Australia. Although herbicides are available for wild oat control, resistance to several commonly used herbicides is becoming more prevalent (12,24). The problem of diminishing weed-control options for wild oat is further compounded by a relatively long seed bank persistence, which ranges up to 9 years depending upon environment, cropping system, and genetics (5). Integrated weed management strategies are needed that couple traditional weed control measures (i.e., tillage and herbicides) with tactics that target the depletion of the soil weed-seed bank.

Persistence of the soil weed-seed bank is regulated by mechanisms that control weed-seed dormancy, maintenance of seed vigor, and resistance to microbial decay (10). Of these three factors, seed resistance to microbial decay has received limited attention by researchers. In principle, physical, chemical, and biological defenses all contribute to seed resistance to decay (15,17,25). Physical defenses include seed coverings, such as the seed coat or the palea/lemma in grass species such as wild oat. Chemical defenses may be in the form of seed exudates or volatile compounds, and consist of a range of sugars, amino acids, and aliphatic and aromatic organic acids, among other compounds (20). Neutral or symbiotic microbial communities associated with weed seeds in the soil can protect seeds from pathogens (6,25) and have been observed in such species as velvetleaf (Abutilon theophrasti L.) (15) and eastern gamma grass (Tripsacum dactyloides L.) (1).

Wild oat has shown little response to fungicides and soil sterilization to reduce potential decay organisms in field studies (11) and inoculation of wild oat with soil microbial communities did not enhance decay (6). However, in other weed species, field inoculations with weed-suppressive microorganisms showed moderately decreased seedling emergence (9,14). In a review by Wagner and Mitchunas (25), a compelling case for microbially mediated weed seed decay is presented, but it is emphasized that considerably more basic research is needed to further understand these processes.

Identifying pathogenic and deleterious microorganisms may lead to crop management strategies that promote weed seed decay and reduce seed longevity, such as altered cultural practices (tillage, rotations, or soil amendments) or by inoculation of soil with deleterious microorganisms (8,11,25). The objectives of this study were: (i) to characerize the mycobiota of wild oat seeds: (ii) screen representative isolates for their seed decay potential; and (iii) investigate the role of soluble hull chemicals as a mechanism of resistance to seed decay. The characterization of these organisms and their interaction with seed chemicals is a necessary step in the development of crop and soil management strategies that promote weed seed decay and/or reduce seed longevity.


Evaluating Caryopsis Decay Potential of Mycobiota in Wild Oat Seed

The no-till plots used in this study were part of a larger tillage/residue study occupying 3000 m² on a Palouse silt loam (silty, mixed mesic, Pachic Ultic Haploxeroll) at the Palouse Conservation Field Station, Albion, WA. Treatments were replicated four times in a block design and each plot was 8 m by 30 m. The wild oat cv. ‘Montana 73,’ a highly dormant wild oat line (19), was grown by Dr. E. P. Fuerst in Pullman, WA from seed collected near Sydney, MT. Seeds of wild oat cv. ‘Montana 73’were mixed with soil from the no-till plots (100 seeds per 100 g soil) and placed in 20 cm × 30 cm mesh bags (washed nylon voile, JoAnn Fabrics, Hudson, OH), sealed, and buried midslope in the plots at a depth of 2.5 cm for six months. Six bags were randomly placed at least 3 m from each other in each of the 4 plots. The seeds were separated from the soil and a subset was then tested for viability (18). The microorganisms colonizing the seeds were isolated by direct plating using half-strength potato dextrose agar (20 g PDA/liter) amended with streptomycin sulfate (8 mg/liter, Sigma, St. Louis, MO) and chlortetracycline (50 mg/liter, Sigma, St. Louis, MO) (23). The seeds were blotted dry with sterile paper towels and hulls were removed aseptically. The caryopses and hulls were surface disinfected by soaking in 70% ethanol (30 sec) and 0.5% sodium hypochlorite (2 min) followed by four rinses in sterile distilled water. They were then blotted dry on sterile paper towels and aseptically bisected longitudinally using a sterile scalpel. Each half was placed on half-strength PDA amended with streptomycin and chlortetracycline (23), with five pieces per plate. The plates were incubated in the dark at 25°C. Plates were observed daily and isolates were transferred onto PDA plates until pure cultures of the isolates were established.

Isolates. Fungal isolates from the buried bag study were identified by morphological characteristics to genus (2) and to species when possible (3,7,20). Pure cultures of Fusarium spp. were grown on carnation leaf agar, PDA, and potassium chloride minimal media to allow formation of typical macro- and microconidia (23). Fungal isolates associated with dormant wild oat (Avena fatua L.) seeds buried for six months in a no-till wheat field were from diverse fungal genera; however, the hull and the caryopses possessed similar fungal species and comparable distribution, and thus are reported together (Table 1). Over 800 fungal isolates were obtained, and the most common species identified were in the genera Papulaspora (32%) or Fusarium (28%). We found some of the same genera as had been previously reported on dormant and non-dormant wild oat lines (13). The isolate species and distribution patterns were the same for the hull and the caryopsis. From those groups, 118 representative fungal isolates were screened for caryopsis decay potential using wild oat ‘Montana 73.’


Table 1. Fungal isolates recovered from wild oat caryopses
and hulls and their frequency of recovery.

Genus and species Percent (%)
Papulaspora sp. 31.9  
Fusarium sp. 27.6  
Mortierella sp. 5.9
Chaetomium globosum 5.3
Ulocladium atrum 5.2
Ulocladium sp. 3.5
Septoria sp. 3.3
Unknown 3.1
Cladosporium malorum 2.6
Phoma medicaginis 1.7
Phoma sp. 1.3
Humicola tainanensis 0.9
Alternaria sp. 0.7
Penicillium sp. 0.7
Fusarium culmorum 0.6
Fusarium merismoides 0.6
Humicola sp. 0.6
Paecilomyces farinosus 0.6
Alternaria alternate 0.4
Humicola grisea 0.4
Trichoderma (koningii) 0.4
Ulocladium dauci 0.4
Cladosporium oxysporum 0.2
Cladosporium sp. 0.4
Mucor circinelloides 0.2
Χ² 78.7  
P < 0.0001

Decay potential. Dehulled wild oat seeds (‘Montana 73’ caryopses) were surface disinfected as described above, transferred to sterile paper towels and air dried in a laminar flow hood. Pathogenicity was tested by placing a 6-mm agar plug from the margins of two-week-old fungal cultures onto the surface of 2% water agar and placing 10 caryopses around the plug. Non-inoculated PDA agar plugs were used as controls. Three replicates were incubated in the dark at 25°C for two weeks to test each of the 118 fungal isolates.

We developed a caryopsis decay rating scale to rank the various stages of decay (Fig. 1). Whole seeds (with hulls intact) of highly dormant wild oat lines were more resistant to decay than non-dormant lines (data not shown). Data were analyzed by nonparametric one-way analysis using the Kruskal-Wallis test (22). We found that only 18 isolates, or 15% of the total tested, elicited caryopsis decay (Table 2; Χ² = 78.7, P < 0.0001). Kremer (16), on the other hand, found higher numbers of decay-producing isolates in his tests of several other weed species. Only six of our isolates decayed wild oat (caryopsis decay rating ≥ 3), and all belonged to the genus Fusarium. Fusarium avenaceum 223a, Fusarium culmorum 637, and Fusarium culmorum 728 all had caryopsis decay ratings of 5.0, the highest value. Decay by F. culmorum 530 was slightly less with a rating of 4.5. F. culmorum 503 and F. culmorum 499 were rated with less decay potential at 3.0. All other isolates decayed wild oat only slightly and were rated at a level of 1.0 or below.


Fig. 1. Wild oat caryopsis decay rating scale.

 

0 − No sign of infection; caryopsis is healthy.

 

1 − Caryopsis tip turning black with mycelial growth on caryopsis surface.


 

2 − Caryopsis tip turning black with brown or black lesions, and mycelial growth on caryopsis surface.

 

3 − Caryopsis tip black or brown with decay starting to advance to the rest of the caryopsis and mycelial growth on caryopsis.


 

4 − Caryopsis tip brown or black with 50% of the caryopsis also turning brown or black and extensive mycelial growth on caryopsis and agar.

 

5 − Entire caryopsis brown or black and extensive mycelial growth on caryopsis and agar.



Table 2. Wild oat caryopsis decay potential of representative fungal isolates from wild oat seed after six months in no-till fields, and percent inhibition of fungal growth by hull extract obtained from water and acetone extraction.

Isolate Caryopsis
decay
rating
x
Water
extract
Acetone
extract
Percent inhibition (%)
Alternaria sp. 358 1.0  2.54 (2)z 1.09 (2)
Chaetomium globosum 644 0.0 5.44 (1) 8.00 (1)
Cladosporium malorum 375 1.0 0.76 (3) 0.77 (3)
Fusarium avenaceum 233a 5.0 3.34 (1) 3.99 (1)
Fusarium culmorum 499 3.0 0.43 (3) 0.49 (3)
Fusarium culmorum 530 4.5 1.37 (2) 1.19 (2)
Fusarium culmorum 637 5.0 7.22 (1) 5.44 (1)
Fusarium culmorum 728 5.0 2.54 (2) 2.54 (2)
Fusarium sp. 503 3.0 1.22 (2) 1.22 (2)
Mortierella sp. 0.7 NDy ND
Mucor sp. 1.0 ND ND
Humicola grisea 592 1.0 3.32 (1) 1.22 (2)
Paecilomyces farinosus 1.0 0.64 (3) 0.74 (3)
Papulaspora sp. 1.0 ND ND
Phoma medicaginis 707 1.0 2.16 (2) 1.07 (2)
Septoria sp. 1.0 ND ND
Ulocladium atrum 585 1.0 19.65 (1)   9.93 (1)
Ulocladium sp. 495 1.0 0.06 (3)   0.001 (3)
Χ² 450 254 273
P < 0.0001 < 0.0001 < 0.0001

 x Average rating based on a 0 to 5 scale (Fig. 1) where 0 = healthy caryopsis; 1 = caryopsis tip turning black, pinhead lesions on caryopsis, mycelial growth on caryopsis surface; 2 = caryopsis tip turning black with brown or black lesions and mycelial growth on caryopsis surface; 3 = caryopsis tip black with decay starting to advance to the rest of the caryopsis, mycelial growth on caryopsis; 4 = caryopsis tip brown or black with 50% of the caryopsis also turning brown or black, extensive mycelial growth on caryopsis and agar; 5 = entire caryopsis turning brown or black, extensive mycelial growth on caryopsis and agar.

 y ND = not determined.

 z Numbers in parentheses indicate ranking.


Over a six month period, many fungal species colonized wild oat seeds that were buried in soil; however, few species of fungi contributed to wild oat caryopsis decay. While the numbers of wild oat caryopses can be higher in no-till systems compared to cultivated soils (4), other factors are also involved in determining seed decay in soil systems and further studies are needed to investigate additional soil communities and their weed suppression (25).

Hull. To determine if the hull was inhibitory against fungal species, hulls of wild oat cv. ‘Montana 73’ (20 g) were ground to pass a 60-m sieve and twice extracted with either water or acetone (5 ml/g dry hulls) (21). This study was repeated three times and all samples were kept at 4°C during extraction, centrifugation or storage. The combined extracts were centrifuged (10,000 × g, 5 min) and the clear supernatant was decanted. The extracts were concentrated in a fume hood and brought to 3 mL total with the appropriate solvent. One mL of the extract was diluted ten-fold and used in the agar-well technique to test antimicrobial activity of the extracts. One-week old cultures of the test fungi were transferred to the center of PDA plates and allowed to grow for 48 h. A 6-mm diameter, flamed cork borer was used to cut four agar wells around the edge of the plate and two plates were used for each treatment. The wells were filled with 50 µl of the water or acetone hull extract. Control wells were filled with sterile water or acetone. The plates were incubated in the dark at 25°C for 5 to 7 days. Zones of inhibition, if present, were measured at 4 equidistant points around the well with a translucent ruler having 1-mm gradations. Percent inhibition was calculated for each extract type { [ (control − extract) / control ] × 100 } and analyzed by nonparametric one-way analysis using the Kruskal-Wallis test and the Wilconox test to rank (22).

Two types of hull extracts, water and acetone, were tested to determine if the extracts had inhibitory activity against fourteen fungal species isolated from wild oat seed (Table 2). Isolates varied in their response to the hull extract (Χ ² = 700, P < 0.0001), but there was no significant difference in response between the two extract types (Χ² = 4.1, P = 0.13). The hull extract exhibited a wide range of activity against the fungal isolates, with inhibition from 0 to 19% compared to growth without the extract. Ulocladium atrum 585 was inhibited 19.7% by the hull water extract and 9.9% by hull acetone extract. F. culmorum 637 was inhibited 7.2% by the water extract and 5.4% by the acetone extract. Chaetomium globosum 644 was inhibited 8% by the acetone extract and 5.4% by the water extract. The growth of four of the fourteen fungal isolates was not affected by the extracts with the hull inhibiting their growth less than one percent. The remaining seven isolates were slightly influenced by the extracts with inhibition ranging from 3.99% to 1.07%. Similar low levels of fungal inhibition were observed in aqueous extracts from seed coats of woodland species where low levels of fungal inhibition were observed in two out of four fungal isolates (27).

Host range. Four Fusarium isolates with the ability to completely decay wild oat caryopses were selected for the host range study (Table 3). The plant species selected were taxonomically diverse and represented broad phylogenetic categories as suggested by Wapshere (26). The four test fungi were grown on PDA for 3 to 5 days at 24°C under near UV and cool white lights on a 12/12-h photoperiod to stimulate sporulation and assist with identification. An agar plug (6 mm) cut from a plate with each of the test fungi was placed on the surface of 2% water agar plates and grown under the same conditions for 5 days. Seeds of different crop and weed species were surface disinfected as above, and eight seeds of each species were arranged radially around the plug. Four plates were used for each isolate and incubated in the dark at 25°C. Seed decay was evaluated one and two weeks after inoculation using the key described in Figure 1. Data were analyzed by nonparametric one-way analysis using the Kruskal-Wallis test (22).


Table 3. Scientific and common names of plant species tested and rating of seed decay by Fusarium avenaceum (F.a.) and Fusarium culmorum (F.c.) in the agar bioassay.

Scientific name Common name F.a.
223a
F.c.
637
F.c.
728
F.c.
530
Monocotyledons Aegilops cylindrica Jointed goatgrass  1.0x 1.0 0.0 0.0
Avena fatua Wild oat 5.0 5.0 4.0 3.0
Avena sativa Oat 5.0 3.0 2.0 3.0
Bromus tectorum Downy brome 5.0 3.0 5.0 2.0
Hordeum vulgare Barley 1.0 1.0 3.0 3.0
Triticum aestivum Wheat 5.0 5.0 3.0 3.0
Dicotyledons Lens culinaris Lentil 1.0 0.0 0.0 1.0
Medicago sativa Alfalfa 1.0 1.0 1.0 0.0
Pisum sativum Pea 2.0 1.0 0.0 0.0

 x Χ² = 345, P < 0.0001


The four Fusarium isolates decayed seeds of wheat (Triticum aestivum L.), oat (Avena sativa L.), and downy brome (Bromus tectorum L.), but were less successful in decaying lentil (Lens culinaris Medik), alfalfa (Medicago sativa L.), pea (Pisum sativum L.), and jointed goatgrass (Aegilops cylindrica Host; Table 3). F. avenaceum 233a, F. culmorum 637 and F. culmorum 728 all decayed wild oat, with F. sp. 530 causing slightly less decay. F. avenaceum 233a was most severe on wild oat, oat, downy brome, and wheat. Wheat was decayed by F. avenaceum 233a and F. culmorum 637 to the greatest extent and F. culmorum 728 and F. culmorum 530 to a lesser extent. F. avenaceum 223a and F. culmorum 728 decayed downy brome more effectively than did F. culmorum 637, and F. culmorum 530 was least inhibitory of all. All the isolates that decayed weeds also decayed one or several crop species, and thus the isolates from this study would not be considered good candidates for use in biocontrol of wild oat.

Only a few fungi isolated from wild oat seed after burial in no-till plots had potential for seed decay of wild oat. Many of the same species found on wild oat occupy the seed of other plant species (1,16,25). This paper identified a method for identifying and evaluating potential seed decay organisms and quantifying stages of decay that can be used for screening of isolates and following decay in the lab or field (Fig. 1). Understanding the progression of seed decay when exposed to various fungi is critical to developing management strategies that reduce the wild oat seed bank. Hull extracts inhibited only a few of the isolates, and only one Fusarium isolate was inhibited by hull extract. Inhibition of the isolates ranged from nearly 20% to no growth inhibition by either extract tested. Only a few of the most prevalent isolates decayed the wild oat caryopses. The fungal strains found to cause the greatest caryopsis decay also decayed other grass species, but only slightly inhibited dicotyledonous species.


Conclusions

Fifteen percent of the isolates tested resulted in decay symptoms on wild oat caryopses. Two genera, Papulaspora and Fusarium, were dominant on wild oat seeds after six months in a no-till field. The hull and the caryopses possessed similar fungal species and similar distribution. Fusarium was the only genera to cause significant decay. A caryopsis decay rating scale was developed that ranged from 0 to 5. Four of the Fusarium isolates had decay ratings greater than four. Only one Fusarium was inhibited by the hull extract. Inhibition by hull extracts, both water and acetone extracts, was low for all other organisms tested, suggesting that soluble chemicals may not play a major role in seed defense from fungi found on or in the seed.


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