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hydrogen sulfide toxicity symptom or autumn decline than soil and water sources. Soil from a field with a history produced symptoms with either well water or chlorinated city water. However, the greenhouse soil with no history of toxicity or autumn decline showed no disease symptoms with the city water. Instead, it showed the autumn decline symptom with the well water.

Introduction

Reports on hydrogen sulfide toxicity or autumn decline, also referred to as akio- chi have increased in recent years from rice fields in Arkansas. Autumn decline often appears in rice fields affected by hydrogen sulfide in anaerobic/flooded conditions. Symptoms include black roots believed to be caused by iron sulfide and root rotting which results from hydrogen sulfide toxicity. The affected rice plants are stunted with yellowish foliage showing up as early as two weeks following the establishment of a permanent flood. The problem is often most severe where cold well water first enters a rice field and may later spread throughout the field, but plants on levees remain healthy. The phenomenon was reported in Arkansas in a limited number of fields in 2004 (Delta Farm Press; Wilson and Cartwright, pers. comm.). However, several more reports of autumn decline occurred across Arkansas from 2012 to 2017. Although the problem may be aggravated in the anaerobic/flooded conditions, there is no clear understanding of why this phenomenon is occurring in different soil types across several rice growing counties in Arkansas. Observations have shown fields having a clay loam soil texture are more prone to the autumn decline phenomenon than other soil textures commonly cropped to rice. The root rotting symptoms often start a few weeks after flood estab- lishment and become progressively worse throughout the season. In situations where root rotting is severe, fungi grow into the crown which limits the function of the whole root system and prevents translocation of water and nutrients from the soil to the plant resulting in crop decline. In moderate to severe cases, tillers break off easily and plant death may occur rapidly leading to significant yield losses. Ongoing field and greenhouse investigations started in 2015 have the following objectives: 1) to search for practical methods to prevent or correct the root blackening and rotting associated with autumn decline; 2) to evaluate the degree of resistance or tolerance of common rice cultivars to autumn decline under greenhouse and field conditions; and 3) to evaluate the effect of soil drainage (the current preventative/rescue strategy) on autumn decline severity and cultivar survival rate.

Procedures

Studies on Strategies to Reduce Hydrogen Sulfide Toxicity and Autumn Decline Field Tests to Evaluate Products in 2017. Spectrum PC and Spectrum PTB were

two formulations of anoxygenic phototrophic bacteria under test. These formulations have been tried since 2015 in both greenhouse and two fields in Woodruff County, Ark.

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In 2017, they were tested again in a field with a history of hydrogen sulfide toxicity and autumn decline at Humphrey, Ark. in a field of cultivar CL153. Similar to the last two seasons, the tests were carried out in micro-plots under field conditions. A half barrel with 2ft diameter and a foot high was fixed into the soil to a depth of 4 inches. The barrels were fixed into the soil a few days before the producer applied pre-flood nitrogen to his field. Nine barrels were used to accommodate three treatments in three replications. The treatments included: Spectrum PC, Spectrum PTB and an untreated control. All barrels were positioned near the bar ditch to allow for easier refilling with water throughout the growing season. All barrels were in a bay in about 6 ft proximity to each other and positioned based on seedling density. The number of seedlings within each barrel showed similar plant density of 35 to 40 plants per barrel area. Treatments were applied a day after the producer’s field was flooded. These micro-plots were checked for refill twice a week maintaining a flood depth of at least 4 inches. Data were collected when the crop reached flowering.

Field Evaluation of Commercial Cultivars for Tolerance in 2017. To evaluate rice

for degree of resistance or tolerance to autumn decline, a test was carried out consisting of 20 commercial cultivars in two replications. The commercial rice cultivars tested in 2016 at Woodruff County were used for comparison in 2017. Gallon sized pots were filled with soil collected from the Humphrey farm that was used for the first experiment. About 10 seeds were planted per pot and were consistently watered from the barrow ditch that was filled with well water. When the rice plants were ready to be flooded, they obtained pre-flood nitrogen and were placed in the barrow ditch. The pots were kept irrigated either by rain or by refilling from the barrow ditch until data collection. At flowering, plants were pulled up carefully from each pot, thoroughly washed to expose the root system, and rated immediately before the blackening disappeared with oxidation. Two rating scales, 0 to 9 for root crown damage and 0 to 5 for root mass discoloration were used as described in the addition-matrix scale developed by Wamishe et al 2018. The highest reading of damage was recorded for the cultivar wherever root crown damage showed variability among sub-samples. In addition, percentage estimates for both root mass discoloration and crown damage were collected. All readings for root crown damage were recorded in reference to crown length (Table 1).

Greenhouse Tests on Effects of Soil/Water Source and Temperature. Soil from

Humphrey, Ark. was collected, at the beginning of October 2017 (approximately a month after rice from the field was harvested). Although top soil with rice stubble was desired, a backhoe gathered more of the deeper subsoil profile. A susceptible rice cul- tivar CL153 was selected based on its field response in 2016 as a susceptible cultivar and planted in gallon sized pots. Eight sets of two pots were prepared to accommodate different combinations of soil types, water sources and water temperatures: Set 1. Field soil + Cold 4 ºC well water; Set 2. Field soil + room 25 ºC well water; Set 3. Field soil + cold 4 ºC Tap water, Set 4. Field soil + room 25 ºC Tap water; Set 5. Greenhouse soil + cold 4 ºC Well water, Set 6. Greenhouse + room 25 ºC well water; Set 7. Greenhouse soil + cold 4 ºC Tap water; Set 8. Greenhouse + room 25 ºC Tap water. The greenhouse soil referred to as silt loam soil, collected presumably from virgin ground, was mixed

with sand and vermiculite peat moss in the ratio of 3:1:1, respectively. The well water used to keep the pots flooded throughout the test period was brought from the field of interest in Humphrey. The well water at 4 ºC was kept in a refrigerator and the tap water was brought down to 4 ºC using ice. The plants were flooded five weeks after planting, NPK fertilizer applied as needed and pots kept flooded for about two months. Due to the frequent overcast days in the winter, six of 1000 watts bulbs were hung over the pots and were tuned on for 9 h from 7.00am to 5.00pm. At boot stage, plants were pulled up, roots washed and percent root mass discoloration were estimated immediately before roots lose their black color due to exposure to atmospheric oxygen. A subset of 10 randomly chosen rice plants were split vertically down the length of the stem and percentage root crown damage was determined.

Results and Discussion

Studies on Strategies to Reduce Hydrogen Sulfide Toxicity and Autumn Decline Field Tests to Evaluate Products. Spectrum PC showed 20% while Spectrum PTB

showed 40% reduction in the number of rice plants with crown rot in micro-plots (half barrels) compared to the untreated control (Fig. 1). These formulations contain anoxy- genic phototrophic bacteria that claimed to consume sulfur in soil solutions in addition to being photosynthetic. No noticeable differences were observed between the treated and untreated rice plants in root mass colors. This was in agreement with the results from previous years’ field and greenhouse tests. There is continued interest in these products for reduction of root crown damage rather than root mass discoloration. Root crown rot has been proven more damaging to crop yield because it is irreversible while root mass discoloration can be reversed by allowing oxygen into rice rhizosphere. By lowering flood depth or using a “drain and dry” strategy for the field helps to replenish depleted oxygen supplies in the soil. This strategy works well for some fields where problems are already known and is used as a protective strategy if done at the right timing to alleviate stress on the plants. However, it may not be a reliable option if the field sizes are too big to drain and re-flood as needed. Moreover, it would be a difficult practice where water resource and pump capacities are limited. Compared to the two fields in Woodruff County for 2016, both hydrogen sulfide toxicity and autumn decline appeared to be more severe at the field in Humphrey, Ark in 2017. In general, after three years of field testing, there is clearly perceived effectiveness of these formulations that varies with different soil types.

Field Evaluation of Commercial Cultivars for Tolerance. A select group of com-

mercial cultivars often included in Producers Rice Evaluation Program (PREP) in rep- licated plots was to be planted at Humphrey, Ark. Due to the narrow planting window caused by the heavy and frequent early season rain, the test was modified to one-gallon pots filled with soil collected from the planned field. Each rice cultivar was replicated twice. About 10 seeds were planted in each pot. After seedlings were well established, pots were kept in the barrow ditch adjacent to the producer’s rice, CL153. Although the cultivars in pots were meant to be treated similar to the adjacent field, the fact that

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they were kept in a barrow ditch in pots with no holes underneath did not allow the cultivars to be equally exposed to the well water like the producer’s rice. Pots did not lose much water so there was less need to refill them. The frequent rain added more water to pots contributing to less well water exposure. When roots were pulled up and evaluated, the maximum root mass discoloration recorded was 60%. Moreover, nearly 75% of the cultivars showed less than 35% root mass discoloration; whereas the CL153 plants in the field showed up to 85% discoloration. Only 55% of the cultivars showed root crown rot in rice plants that ranged from 5% to 30%. Using the 0 to 9 scale, root crown rotting ranged from 0.5 to 9 and all cultivars showed root mass discoloration (Fig. 2). Root mass discoloration is often considered less damaging to the crop yield than root crown damage. Based on the matrix-addition scale developed by Wamishe et al., 2018, cultivars that showed root crown rot above 30% are categorized as intolerant/ susceptible regardless of the percentage of root mass discoloration (Fig. 2).

Greenhouse Tests on Effects of Soil/Water Source and Water Temperature. Since

the cultivar tolerance screening in section 2 above rendered less intensity of root mass discoloration and root crown damage, it raised questions on sources of impact factors -- whether it is the water, the soil, a combination of the two or water temperature which caused the mild symptoms in the pot experiment. Because the pots were kept flooded by rain, more than by well water and also more from barrow ditch than from well direct, designing a test that separates and shows the impact of the water source was deemed necessary. A greenhouse test was designed consisting of 8 sets of treatments in two replications. Soil and water were collected from the field in Humphrey, Ark. The eight treatment comprised the following combinations: Field soil + cold 4 ºC well water; Field soil + room temp well water; Field soil + cold 4 ºC Tap water; Field soil + room temp Tap water; Greenhouse soil + cold 4 ºC well water; Greenhouse + room temp well water; Greenhouse soil + cold 4 ºC Tap water; Greenhouse + room temp Tap water. The greenhouse soil was mixed in a ratio of 3:1:1 soil: sand: Vermiculite peat moss. The greenhouse soil was known to have no history of hydrogen sulfide toxicity or autumn decline.

When roots were examined, they were not as black as expected. This was likely due to excessive loss of water particularly during weekends from the heat coming from the light bulbs. The 1000 watts bulbs were hung over the pots to simulate the summer field light intensity for 9 hrs. Unless pots are kept full of water, the oxidation process can occur which reverses reduction to hydrogen sulfide and FeS. The FeS is known to be responsible for coating the root system black.

Although root mass blackening was not as intense as expected, blackness in crown root bases and root crown browning were evaluated for symptoms of autumn decline that may have followed from hydrogen sulfide toxicity in the course of flood treatment. As shown in Table 2, saturated soil flooded with well water at room temperature or 4 ºC treatments showed more than 80% of the plants with a black root crown base. Among these, up to 27% of the rice plants had damaged root crowns. Contrarily, none of rice plants in pots treated with greenhouse soil and flooded with tap water at either temperature showed any symptom of crown base discoloration or root crown rotting. On the other hand, rice plants in pots treated with field soil and flooded with tap water showed positive symptoms to demonstrate that the soil alone could cause the symptoms

even if the water source was different. The most interesting result from this test was that the well water and greenhouse soil combination showing the symptoms of root crown base blackening with nearly 4% of the plants showing root crown damage. The effect of water temperature in this or other treatments was erratic and appeared to have less impact (Table 2). Although this test is preliminary, it needs to be repeated to definitively determine the role of each treatment factor either alone or in combination.

Significance of Findings

Every year, from Arkansas rice production fields, more reports on root blacken- ing and crown rotting associated with hydrogen sulfide toxicity and autumn decline have occurred. In some fields, draining surface flooded water improved the situation. However, in other fields the “drain and dry” approach did not improve the situation enough to salvage the crop. A better understanding of this problem and alternative ways of managing the problem in various soil types would permit growers to make the best decisions possible to avoid losses due to the failure of the “drain and dry” strategy. Additionally, the “drain and dry” approach does not work if a field is not in a manageable size because of water resource and pump capacity. Knowledge of cultivars susceptibility/intolerance and the discovery of additional management options could prevent significant losses that have occurred to some rice fields.

Acknowledgments

The extension rice pathology program appreciates the funding and support from, the Arkansas rice producers through monies administered by the Arkansas Rice Research and Promotion Board, and the University of Arkansas System Division of Agriculture. We are grateful to Rick Cartwright of the University of Arkansas Division of Agriculture for his technical help. Our appreciation goes to rice producers that allowed us to use land and soil to research. We do appreciate the Arkansas County agents and consultants who are interested in the study and helping in their capacity. We thank Grant Beckwith for his continual interest in the area and Anita Kelly for her interest and help.

Literature Cited

Delta Farm Press Sept 24, 2004 at http://deltafarmpress.com/rice-malady-identified- hydrogen-sulfide-toxicity.

Dobermann, A. and T. Fairhurst. 2000. Rice: Nutrient disorders and nutrient manage- ment. Sulfide Toxicity. Oxford Graphic Printers Pte LTD. PP126-128.

Wamishe YA, Hardke J, Mulaw T, Gebremariam T, Belmar S, Kelsey C1 , Roberts, T and Fryer J. 2018 . A Method to Estimate Field Response to Hydrogen Sulfide Toxicity and Autumn Decline in Rice Cultivars. J Soil Sci Plant Health 2:2.

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Table 1. Rating scales used to rate crown discoloration and root discoloration in cultivars grown in soil with history of autumn

decline/hydrogen sulfide toxicity in Ark.

Rating Crown Length Discoloreda _{Rating Blackened}Root Mass a

(0-9 scale) (%) (0-5 scale) (%)

0 0 0 Clean as in levee roots

1 10 1 10 2 20 2 25 3 30 3 50 4 40 4 75 5 50 5 75 or > 6 60 7 70 8 80 9 90 or >

a _{Roots need to be washed well and rated immediately, up to 10 root}

crowns need to be examined. Numbers shown under % columns refer to range of estimate.

For instance: 10 refers to discoloration percentage > 0 =10.

Table 2. Effect of soil source, water source, and temperature on the severity of autumn decline in the greenhouse.

Soil Water Water Plants with Plants with source source temperature black crown base crown rot

---%---

Field Well 4 ºC 83 5

Field Well room 88 27

Field Tap 4 ºC 64 50

Field Tap room 100 4

GH Well 4 ºC 83 0

GH Well room 100 4

GH Tap 4 ºC 0 0

GH Tap room 0 0

GH: Greenhouse; Well: Well water from Humphrey and Tap: chlorinated city water; Room: room temperature either of the lab or the greenhouse.

Fig. 1. Effects of two bio-formulations in reducing crown rot in rice plants grown in a field with a history of hydrogen sulfide toxicity and autumn decline. Note: plants in barrow

ditches often are highly affected.

Fig. 2. Percentage root mass discolored, plants with crown rot and extent of crown rot as estimated in 0-9 scale in a field test using pots filled with soil with a history of hydrogen

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