1. Introduction
Sipha maydis Passerini is distributed throughout most cereal producing countries of the world. In Pakistan [1] and Iran [2], S. maydis is reported to be among the most common aphid species affecting wheat. S. maydis was first reported in Argentina in 2002 followed by spread throughout the country by 2006 where it primarily infested wheat, barley, and wild grasses [3]. By 2007 it was detected in the US infesting giant wild rye, Leymus condensatus in CA [4]. Infrequent reports of S. maydis on wild grasses and wheat ranged from the SW US to the SE costal states over the next few years. A report of S. maydis infesting oats near Albuquerque, NM in 2014 instigated a survey in CO in 2015 where it was found on winter annual grasses at many sites in WC and SW CO [5]. The common name, hedgehog grain aphid, HGA, was suggested for this new invasive pest. HGA has been reported on volunteer wheat in UT. Multi-state surveys from 2015-2017 in the Rocky Mountain and Southern plains states determined HGA was broadly distributed and adapted to a variety of wild grasses, barley, sorghum, and wheat with the greatest concentration in W CO and NW NM [6].
HGA feeding was devastating to mature wheat plants on the prairies of Buenos Aries province in Argentina in 2005 [3]. HGA was found in young leaves in the fall and in the ligula area of flag leaves in the late spring where chlorosis reduced functional leaf area and inhibited head growth. Chlorosis from HGA feeding spread beyond the feeding site indicating HGA is a phytotoxic aphid. HGA is also an efficient vector of the luteovirus barley yellow dwarf (BYDV) a significant disease of wheat and barley [7]. HGA is a dual threat to small grain production.
Barley production in the Great Plains and Rocky Mountain States was valued at $529 million in 2018 [8]. Development of resistant cultivars is the most economically and environmentally sound control method for insect attack. Few studies on resistance to HGA have been reported, and in 2013 leaf pubescence of several wheat genotypes was determined as not an effective for providing resistance against S. maydis [9]. However, in 2011 antibiosis was evaluated in 47 commercial wheat cultivars from Argentina and 24% had high levels of antibiosis with both nymphal development and reproduction negatively affected [10]. The vulnerability of US cultivars to HGA has yet to be established. Identification of HGA resistance is inhibited by the physical restraints of quarantine required by APHIS to experiment with HGA in the US. Cross-resistance, where sources resistant to one aphid species is also resistant to another species, has been demonstrated in wheat [11] and sorghum [12]. Identification of cross resistance for HGA in established aphid resistant barley cultivars would present a tremendous opportunity to quickly deploy resistance to HGA. There are 2 greenbug resistance genes, Rsg1 and Rsg2, and 3 RWA resistance genes, Rdn1, Rdn2, and Rdn3, identified in barley. These genes are available in 5 deployed cultivars where Rsg1 = Post 90, Rsg2 = STARS 1501B barley, and an experimental barley 00BX 11-115 which includes Rsg1 and an unidentified resistant source known as Rdny (Table 1). The known resistance RWA resistance genes Rdn1, Rdn2, and Rdn3 are in STARS 9301B, while Rdn1, Rdn2 are also in STARS 9577B. A 6th cultivar cv Mesa has been deployed with a unique, unidentified source of RWA-resistance termed Rdnx. (Table 1). It is the objective of this study to determine the potential for cross resistance to HGA by measuring antixenosis (plant preference), plant damage, plant growth, and intrinsic rate of increase in cultivars/germplasm previously identified as resistant to GB and RWA.
2. Materials and Methods
HGA used in this study came from a S. maydis colony collected near Taos, NM in 2017. They were cultured on aphid susceptible “Yuma” wheat in clear plastic cylinder cages in a room with a temperature of 21˚C and a photoperiod of 14:10 L:D provided by a light rack equipped with eight, fluorescent grow lights (#7866113, Philips Inc., Guadalajara, Mexico).
Aphid-resistant cultivars and germplasm utilized in this study include Post 90, STARS 1501B, STARS 9301B, STARS 9577B, Mesa, and experimental line 00BX 11-115. Resistant genes and other agronomic characteristics for each entry are found in Table 1. Rsg1 has been deployed in cultivar “Post 90” [13], while Rsg2 has been released in germplasm line STARS 1501B [14]. Four Russian wheat aphid-resistant barley cultivars have derived their resistance from 2 germplasm lines STARS 9301B [15] and STARS 9577B [16]. STARS 9301B carries 3 RWA-resistant genes, Rdn1, Rdn2, and Rdn3 [17], while STARS 9577B carries Rdn1 and Rdn2 [18]. The variety Mesa carries unnamed resistance gene(s) from another source [19]. Experimental line 00BX 11-115 has both Rsg1 resistance as well as RWA resistance from a unique source. Cultivars Morex [20] and Schuyler [21] were susceptible controls. Both spring and winter growth habits are represented by the cultivars/germplasm (Table 1).
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Table 1. Resistance genes, target aphid, growth type and designation of barley entries.
*Rdnx and Rdny indicate a resistance gene not yet identified.
Plant damage response and aphid preference were measured in a completely randomized design with 10 replications in the fall of 2018. The experiment was conducted in a growth chamber (Conviron®, Winnipeg, Canada) set at 21˚C and 14:10 L:D photoperiod with lighting provided by seven TS 32W Ecolux® daylight fluorescent lamps (Fairfield, Connecticut, USA) and four 60W incandescent bulbs. This model of growth chamber is divided in two identical sections, and within these two were conducted an infested portion where entries were challenged with HGA, while the other compartment grew an identical set of entries that were not infested allowing for comparative plant growth measurements such as numbers of leaves and plant height. Each of the entries were planted 2 seeds to a cell in potting soil (Sun Gro Horticulture, Agawam, MA 01001 U. S.) within 25 cell miniflats (Growers Supply, Dyersville, IA 52042). The Mini-flats measured 18 cm in width × 27 cm in length × 30 cm in depth were then placed in a tray measuring 27 cm wide × 38 cm long × 6 cm high filled with construction grade sand which served as a bed to seal a metal frame cage over each mini-flat. Metal frame cages measuring 30 cm wide × 40 cm long × 30 cm tall and were covered with fine mesh nylon screen. Seven d after planting, the healthiest seedling of the 2 planted was chosen to continue in the experiment, while the other was removed. When seedlings were 4 - 6 cm in height ten d after planting, they were infested with 10 aphids on each entry by transferring them one at a time using a camel-hair brush. A companion set of non-infested controls was planted and kept aphid free in an identical compartment of the growth chamber as described above. Five days after infesting, the number of sugarcane aphids per seedling was counted to determine aphid preference, antixenosis by removing the caged flat from the growth camber and removing the screened framed cage from the tray where each plant was examined non-destructively and HGA counted using a 10x magnifying visor. On the day of the evaluation, 17 d after infesting, and when the susceptible entries were near death, plant damage was assessed using the 1 - 9 rating scale described for RWA in which ratings increase with increasing levels of leaf chlorosis/necrosis (1 = healthy, 2 = 1% - 5% chlorosis, 3 = 5% - 20% chlorosis, 4 = 21% - 35% chlorosis, 5 = 36% - 50% chlorosis, 6 = 51% - 66% chlorosis, 7 = 66% - 80% chlorosis, 8 = 81% - 95% chlorosis, and, 9 = dead; [22]. The number true leaves were counted excluding the cotyledon leaf and plant height was taken using a metric ruler where plant height for each entry and replication were measured from the soil surface to the most distal portion of the top leaf. Differences in the number of true leaves and plant height within an entry were made by subtracting each replication of the infested entries from the non-infested replicates.
Intrinsic rate of increase parameters was measured in the same growth chamber described above with a temperature of 22˚C and a 14:10 L:D photoperiod. Two seeds of each barley entry were planted into potting mix described above within a 7.5 cm plastic pot. Each individual pot was covered with a 5 cm diameter × 41 cm plastic tube cage and pots placed in the growth chamber. When seedlings reached the 2-leaf stage they were infested by a single viviparous female which was removed after 24 h. From these nymphs on each entry, a single, 24 hold, nymph per seedling was selected to remain on the plant where the development time to reproductive adult (d) and reproduction (Md) was recorded. Intrisic rate of increase (rm) was calculated using the formula: rm = 0.0738 (1ogeMd)/d [23].
Data on antixenosis, reduction in plant height and leaf number were analyzed using PROC MIXED [24] where mean comparisons were made by using the Least Significant Differences Method (LSD) at P > ltl ≤ 0.05 level [24]. Plant damage ratings for barley entries were compared by nonparametric analysis (NPAR1WAY) using Wilcoxon tests to conduct pair-wise comparisons among all entries to establish significant differences at P > X2 ≤ 0.05. Aphid antixenosis and plant growth factors were correlated to leaf damage using Pearson’s correlation coefficients (Prob > lrl under H0: Rho = 0) using PROC CORR [24].
3. Results
There were varying degrees of damage caused by HGA feeding on the 6 barley genotypes that had previously been proven resistant to either GB or RWA, and the 2 known sources that were used as susceptibles in the evaluation (Figure 1) The entries barley 00BX 11-115 and Post 90 had significantly fewer aphid numbers per seedling (Table 2) than any other entry showing non-preference (high antixenosis) by HGA compared to all other entries. Morex and STARS 9301B had aphid numbers significantly greater than all other entries indicating low antixenosis for HGA. All other entries had intermediate aphid numbers/seedling. Antixenosis was only moderately correlated with seedling damage ratings (r = 0.42. P = 0.0001; n = 80).
00BX 11-115 and Post 90 had significantly lower plant damage ratings than all other entries (Table 2). Their low damage ratings indicate a high level of resistance in these lines. All other entries had a moderately susceptible to susceptible level of resistance to HGA.
HGA infestation reduced seedling height on all barley entries by 47.9% - 61.9% in comparison to non-infested controls. 00BX 11-115 had the least reduction but was not significantly different than reductions for Post 90, STARS 1501B and Morex. There was a poor relationship between plant damage rating and percent reduction in seedling height (r = 0.24. P = 0.03; n = 80).
Percent reduction in leaf number of entries for infested seedlings compared to non-infested controls ranged from 26.7% - 33.3% and was not significant (Table 2). There was no significant relationship between percent leaf number reduction and plant damage ratings (r = 0.18. P = 0.10; n = 80) or between percent leaf number reduction and percent seedling height reduction (r = 0.12. P = 0.29; n = 80).
Days to reproductive adult, fecundity rate, and intrinsic rates of increase were significantly affected by barley entry (Table 3). HGA feeding on Post 90 had the greatest number of days from newborn nymph to reproductive adult and was significantly greater than all lines but 00BX 11-115 and STARS 9301B. 00BX 11-115 had significantly greater number of days to reproduction than all lines except Post 90, STARS 9301B, and Schuyler. The number of days to reproduction of HGA feeding on STARS 9301B was significantly greater than the number of days for remaining lines except Schuyler. Fecundity rate was significantly lower for 00BX 11-115 and Post 90 than all other entries. Intrinsic rate of increase was significantly lower for 00BX 11-115 and Post 90. No aphid mortalities occurred on any of the entries during the study.
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Figure 1. Images of damage rating results from HGA feeding on resistant and susceptible barley entries. The damage rating scale was 1 - 9 where 1 is a healthy plant showing no chlorosis and 9 is a dead chlorotic plant (Burd et al., 1993).
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Table 2. Antixenosis, plant damage rating and percent reductions in seedling height and leaf number.
* Means within a column followed by a different lower-case letter are significantly different (P ≤ 0.05). **1 - 3 = highly resistant, 4 - 6 = moderately resistant to moderately resistant, and 7 - 9 = susceptible.
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Table 3. Effects of entry on HGA intrinsic rates of increase (rm) and its components.
* Means within a column followed by a different lower-case letter are significantly different (P ≤ 0.05).
4. Discussion
Significant and substantial differences in plant damage response among entries occurred when challenged by HGA infestation. 00BX 11-115 and Post 90 were the only entries which expressed strong resistance to feeding damage while other entries suffered much higher plant damage that clearly reflected susceptibility to HGA (Table 1, Figure 1). 00BX 11-115 and Post 90 also expressed high levels of antixenosis and antibiosis (rm) in comparison to susceptible entries. Closer examination of HGA feeding damage after six days on susceptible Morex revealed numerous feeding sites with dark necrosis and extensive chlorosis. These distinctive types of feeding damage have also been described for another phytotoxic cereal aphid, the greenbug, feeding on wheat [25] [26]. In contrast, feeding sites on resistant Post 90 had very limited necrotic lesioning with adjacent cell bleaching and limited chlorosis (Figure 1). Post 90 is the greenbug resistant parent of 00BX 11-115. They share the single dominant greenbug resistant gene, Rsg1, which suggests this gene may be responsible for cross-resistance to HGA. Not all greenbug resistance genes are cross-resistant to HGA. STARS 1501B which carries the other identified single dominant greenbug resistance gene, Rsg2, was susceptible to feeding damage and was among the entries with the lowest levels of antixenosis and antibiosis.
Greenhouse seedling resistance ratings have been shown to accurately predict field resistance in terms of grain yield for RWA [27]. In 2007 [3] reported HGA in the ligula area of flag leaves in the late spring where chlorosis reduced functional leaf area and inhibited head growth. Although substantial reductions in seedling height and leaf number occurred in all entries, these kinds of reductions due to severe infestation levels in caged plant trials are common regardless of resistance or susceptible status [13] [22] [28], however 00BX 11-115 and Post 90 withstood high infestations in the caged conditions of this study and remained green and only slightly damaged while the other entries sustained severe chlorosis and were near death. Future studies need to be conducted to establish that resistance in 00BX 11-115 and Post 90 persists throughout the life of the plant and offers meaningful resistance in terms of grain yield in the field.
In 2019, [6] reported that HGA is currently present in low numbers in the Rocky Mountain and Southern Plains regions of the US and suggests that it is in the early stages of adaptation to these areas. It was also reported that there is a general lack of predators and parasites that could naturally help regulate HGA. Development of resistant cultivars would be the most economically and environmentally sound defense against this newly introduced pest. Cross-resistance identified in greenbug resistant sorghum [12] has been utilized to immediately deploy cultivars and fast track development of new resistant cultivars to a recently introduced pest of sorghum, the sugarcane aphid. Identification of cross-resistance in 00BX 11-115 (soon to be released as the cultivar Fortress) and Post 90 provides an opportunity to quickly deploy and develop cultivars resistant to HGA.
5. Conclusion
From the eight genotypes of which 6 were known to have resistance to either RWA or GB, only the Post 90 which has the Rsg1 resistance gene, and 00BX 11-115 which has both Rsg1 and an undetermined Rdny are known to be resistant to both GB and RWA. We determined in this experiment that Post 90 and 00BX 11-115 were also resistant to the HGA. Discovery of these sources of resistance will allow time for the further development and deployment of more resistant sources of barley to provide growers with resistant genotypes that will prevent loss in yield and perhaps decrease the chance for the spread of barley yellow dwarf virus, the most devastating viral disease in barley. The economics of having resistant aphid sources for this particular aphid species infesting barley has not determined however numerous examples of significant positive economic impacts of providing resistant cereal grains against virulent cereal aphids exist.
Acknowledgements
We thank Melissa Pals, USDA-ARS, Stillwater, Oklahoma, for her technical expertise in maintaining aphid colonies used in this study. The U.S. Department of Agriculture (USDA) prohibits discrimination in all its programs and activities on the basis of race, color, national origin, age, disability, and where applicable, sex, marital status, familial status, parental status, religion, sexual orientation, genetic information, political beliefs, reprisal, or because all or part of an individual’s income is derived from any public assistance program. (Not all prohibited bases apply to all programs.) Persons with disabilities who require alternative means for communication of program information (Braille, large print, audiotape, etc.) should contact USDA’s TARGET Center at (202) 720-2600 (voice and TDD). To file a complaint of discrimination, write to USDA, Director, Office of Civil Rights, 1400 Independence Avenue, S.W., Washington, C.C. 20250-9410, or call (800) 795-3272 (voice) or (202) 720-6382 (TDD). USDA is an equal opportunity provider and employer.
Abbreviations
d, day;
L:D, light: dark;
rm, intrinsic rates of increase;
RWA1, Russian wheat aphid Biotype 1;
RWA2, Russian wheat aphid Biotype 2.