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2009. Plant Management Network. This article is in the public domain.
Accepted for publication 13 April 2009. Published 9 June 2009.


Nitrogen Fertilizer Affects the Severity of Anthracnose Crown Rot Disease of Greenhouse Grown Strawberries


Barbara J. Smith, Research Plant Pathologist, USDA-ARS, Thad Cochran Southern Horticultural Laboratory, Small Fruit Research Unit, P.O. Box 287, Poplarville, MS 39470


Corresponding author: Barbara J. Smith. Barbara.Smith@ars.usda.gov


Smith, B. J. 2009. Nitrogen fertilizer affects the severity of anthracnose crown rot disease of greenhouse grown strawberries. Online. Plant Health Progress doi:10.1094/PHP-2009-0609-01-RS.


Abstract

The influence of nitrogen, phosphorus, and potassium on the severity of anthracnose crown rot was evaluated in three greenhouse studies. Strawberry plants were fertilized three times weekly with a modified Hoagland's Nutrient Solution containing the treatments and inoculated eight weeks after treatment applications began with a conidial suspension of the causal pathogen, Colletotrichum fragariae. Disease severity was rated 30 days later on a scale of 0 (no symptoms) to 6 (plant dead). In the first study, the effect of N, P, and K levels was evaluated using 16 treatments: eight N levels with either low P and K or high P and K. Disease severity ratings and percent foliar N increased as N level increased but were not influenced by the level of P and K. In two other experiments, seven N sources were each evaluated at three levels. Plants receiving 160 ppm N had higher disease severity ratings than plants receiving 0 or 40 ppm N. Among plants receiving 160 ppm N, those treated with Ca(NO3)2 had the least disease. When N fertilizer is applied to strawberry plants as Ca(NO3)2, anthracnose crown rot severity should be less severe than when N is applied in ammonium forms.


Introduction

Anthracnose crown rot, caused by the fungus, Colletotrichum fragariae, may infect all above-ground parts of the strawberry plant (5,7). Petiole and stolon infections are characterized by dark sunken lesions, and leaf infections are characterized by small black leaf spots. When the fungus infects the crown, wilt and rapid plant death often follows. Internal symptom of a crown infection is a red discoloration of the crown usually extending in a “V” shape from the side of the crown in towards the center. Colletotrichum gloeosporioides and C. acutatum also cause anthracnose diseases of strawberry (5,7). In the southeastern United States, anthracnose crown rot and fruit rot can be severe in strawberry production fields where Colletotrichum spp. often spread rapidly during wet harvest seasons, sometimes causing a total crop loss. Fungicides are used to control anthracnose diseases (13), but several are no longer effective due to the development of fungicide tolerant strains of the pathogens (10).

Field observations in Florida showed that growing strawberry plants in soils with low N levels reduced the severity of anthracnose (5), but fruit production was also severely reduced. Nitrogen form and calcium level are both known to influence disease severity (12). For example, calcium has been used to control soil-borne diseases caused by Pythium spp. on various crops (6), and nitrate forms of N were shown to suppress Fusarium wilt in tomato whereas ammonium forms increased disease severity (15). However, the effect of N on disease management varies with the disease. Ammonium sulfate was shown to be more suppressive of black root rot of strawberry than Ca(NO3)2 (2). The objectives of these studies were to determine (i) the influence of N, P, and K levels; and (ii) the influence of N source and concentration on the severity of anthracnose crown rot.


Fungal Pathogen and Host Strawberry Plants

Cultures of C. fragariae isolate CF-63 (9) were initiated from silica gel cultures maintained at USDA-ARS in Poplarville, MS, and grown on potato dextrose agar:oatmeal agar (1:1, v:v) under continuous fluorescent light at room temperature (20 to 28°C). Conidial suspensions used for inoculations were prepared from 7 to 14-day-old cultures. Inoculum was prepared by flooding each culture plate with sterile deionized water and gently scraping the agar surface with a glass rod to remove conidia. The resulting conidial suspension was filtered through cheesecloth, and the final conidial suspension was adjusted to a concentration of 1.5 × 106 conidia/mL. A hand pump sprayer was used to apply the inoculum as a mist uniformly over the foliage of the plants to the point of runoff. Inoculated plants were immediately placed in a dew chamber at 100% relative humidity and 30°C for 48 h and then into a greenhouse for 5 weeks with a temperature of 22 ± 7°C.

Plants of the strawberry cultivar (Tangi) and four clones from the USDA-ARS breeding program [MSUS 37, MSUS 74, MSUS 70, MSUS 98 (3,8)] were grown in pasteurized sand with one plant per 10-cm plastic pot in a greenhouse (20 ± 7°C). Within each experiment, plants of each strawberry clone were randomly assigned to N treatment. Test nutrients were prepared in a modified Hoagland's Nutrient Solution (4) containing (NH4)2SO4 or NH4NO3, NaH2PO4.H2O, KCl, CaCl2.2H2O, MgSO4.7H2O, Fe as Fe-chelate, MnCl2.4H2O, ZnSO4.7H2O, CuSO4.5H2O, H3BO3, CoCl2.6H2O, and MoO3. Fifty ml of a treatment solution was applied three times weekly to each pot. Pots were watered with tap water on alternate days and flushed with excess water once a week. Eight weeks after the initiation of treatment applications, all plants in an experiment were inoculated with a suspension of C. fragariae conidia, incubated in a dew chamber for 48 h, and then held in the greenhouse.

Disease severity rating. Inoculated plants were evaluated for disease severity 30 days after inoculation on a scale of 0 (no symptoms) to 6 (plant dead) (9). Rating categories were: 0 = plant with no visible lesions, 1 = plant with petiole lesions < 3 mm long, 2 = plant with petiole lesions 3 to 10 mm long, 3 = plant with petiole lesions > 10 to 20 mm long, 4 = plant with petiole lesions > 20 mm long, 5 = plant whose youngest leaf wilted with or without petiole lesions, and 6 = dead plant with necrotic crown. An average disease rating of 2.0 or less was considered a resistant response, an average rating of 4.0 or greater was considered a susceptible response, and an average rating between 2.1 and 3.9 was considered an intermediate response.


Effect of Nitrogen, Phosphorus, and Potassium Levels

The effect of N, P, and K levels was evaluated using 16 treatments. Eight N levels (0, 5, 10, 20, 40, 80, 160, and 320 ppm N) were applied as NH4NO3 with either low P and K (8.7 ppm P and 16.6 ppm K) or high P and K (35.8 ppm P and 66.4 ppm K). Phosphorus was applied as NaH2PO4 and K was applied as KCl. The design was a randomized complete block with five one plant replications of each of three strawberry clones (MSUS 37, MSUS 70, and MSUS 98). After five weeks of nutrient solution application, the photosynthesis rate of each plant was measured on the most recently fully expanded leaf using a portable photosynthesis system (LI-6400, Li-Cor Co. Ltd., Lincoln, NE) equipped with an infrared gas analyzer. The fourth and fifth leaves were collected from all plants, dried at 70°C, and analyzed for percent foliar N by micro-Kjeldahl.


Effect of Nitrogen Source and Level

Seven N sources [(NH4)2SO4, NH4Cl, (NH4)2HPO4, NH4NO3, NaNO3, KNO3, Ca(NO3)2] were evaluated at three levels (0, 40, and 160 ppm N) on three strawberry clones (MSUS 37, MSUS 70, and MSUS 98) with five replications in Nitrogen Source Study I. The same seven N sources were evaluated at three levels (0, 40, and 120 ppm N) on four strawberry clones (Tangi, MSUS 70, MSUS 74, and MSUS 98) with four replications in Nitrogen Source Study II.


Data analysis

For all analyses, disease severity data were first transformed by adding 0.5 to the disease severity rating and then calculating the square root. Statistical analyses were performed with a SAS package (Release 6.07, 1989; SAS Institute Inc., Cary, NC). When the F values were significant, mean comparisons were determined by least significant difference value at P = 0.05. Regression analysis was used to determine the relationship between N treatment levels and disease ratings, foliar N content, and photosynthesis rates.


Effect of Nitrogen, Phosphorus, and Potassium Levels

Disease ratings. Disease severity ratings increased as treatment N levels increased (Table 1, Fig. 1). When averaged across all treatments, ‘MSUS 37’ plants had higher disease severity ratings than ‘MSUS 70’ and ‘MSUS 98’ plants (Table 2), but all three were in the intermediately susceptible range. When averaged across the three strawberry clones, the highest disease severity ratings were from plants receiving the highest levels of N and the lowest scores were from plants receiving 40 ppm N or less. The factors (P and K levels, interaction between P and K levels and N level, and interaction between strawberry clone and fertility treatment) were not significant.


 

Fig. 1. Relationship between treatment N levels and disease rating, photosynthesis rate, and percent foliar N of three strawberry clones following inoculation with Colletotrichum fragariae.

 

Table 1. Results of regression analysis between eight treatment N levels (NLEL) with either low or high P and K levels (PKLEV) and percent foliar N (% FN), disease ratings (DR), and photosynthesis rate of greenhouse grown strawberry plants following inoculation with Colletotrichum fragariae. Note: NLEV = 0 and PKLEV = 0 were not included in the analysis when N levels were evaluated.

            DR % FN Photosynthesis
R2 0.46 0.73 0.51
Intercept 1.24 1.62 0.26
Pr>F
      Clone 0.0001 0.0001 ns
      PKLEV ns ns ns
      NLEV 0.0001 0.001 0.0001

Table 2. Effect of N level applied with either low or high levels of P and K to strawberry plants grown in sand culture in the greenhouse on the anthracnose crown rot disease ratings (DR) and percent foliar N (% FN). Means are of five plants each of three strawberry clones (MSUS 37, MSUS 70, and MSUS 98).

N levelv DRw % FNx Clone DR % FN
0 ppm      2.3cy      1.24f MSUS 37 3.8a 1.57b
5 ppm      2.4c      1.46de MSUS 98 3.0b 1.81a
10 ppm      2.4c      1.42e MSUS 70 3.3b 1.57b
20 ppm      2.9c      1.64c P and K levelz
40 ppm      2.9c      1.52d
80 ppm      3.9b      2.03b
160 ppm      5.1a      2.05b Low 3.5 1.84
320 ppm      5.6a      2.93a High 3.4 1.78

 v Each treatment solution also contained Ca, Mg, Fe, S, Mn, Zn, Cu, B, Co, and Mo. The pH of each solution was adjusted to 6.5 with 1N HCl or 1N NaOH.

 w Disease severity rated on a scale of 0 = no symptoms to 6 = plant dead 30 days after inoculation with Colletotrichum fragariae isolate CF-63.

 x % Foliar N determined by micro-Kjeldahl.

 y Mean separation within columns within factors by Least Significant Difference, P = 0.05.

 z Low P and K (P = 8.7 ppm; K = 16.6 ppm); high P and K (P = 35.8 ppm; K = 66.4 ppm).


Foliar nitrogen level. Foliar N increased as treatment N levels increased (Table 1; Fig. 1), but there was no effect on foliar N levels due to P and K levels. There was a significant difference in foliar N level among the strawberry clones: the average foliar N content of ‘MSUS 98’ plants was significantly higher than that of ‘MSUS 70’ and ‘MSUS 37’ plants (Table 2). When averaged across the three strawberry clones, the highest levels of foliar N were obtained from plants treated with 320, 160, or 80 ppm N, and the lowest levels were from plants receiving 0, 5, or 10 ppm N.

Nitrogen levels of 2.0 to 3.0% are generally considered adequate for normal growth and development of strawberry plants (1) while plants with N levels < 2.0% are considered deficient. In these studies plants whose leaves contained < 1.7% foliar N at inoculation had lower disease ratings than plants with greater levels of foliar N (Table 3). Disease ratings of all three clones were in the susceptible range when sufficient N was applied to achieve adequate (i.e., > 2%) levels of foliar N (Fig. 1). ‘MSUS 37’ plants received a disease severity rating > 4.0 (in the susceptible range) at the treatment N level of 60 ppm and foliar N content of 1.5%, ‘MSUS 70’ plants received a disease severity rating > 4.0 at the treatment N level of 100 ppm and foliar N content of 1.8%, and ‘MSUS 98’ plants received a disease severity rating >4.0 at the treatment N level of 120 ppm and foliar N content of 2.0%.


Table 3. Average disease severity ratings (DR) at
each foliar N levelw following the application of nutrient
solutions containing various levels of N to strawberry
plants grown in sand culture in the greenhouse.

Foliar N (%)x DRy
1.0  2.4 cz
1.1 2.2 c
1.2 2.6 c
1.3 2.9 c
1.4 2.3 c
1.5 2.7 c
1.6 2.9 c
1.7 2.6 c
1.8 4.7 b
1.9 4.6 b
2.1 4.8 b
2.3 4.5 b
2.5 6.0 a
2.8   5.6 ab
2.9   5.3 ab
3.0 4.6 b

 w Disease severity rating averaged across any of the
16 nutrient treatments and three strawberry clones
(MSUS 37, MSUS 70, and MSUS 98) whose % foliar N
fell into each group. Each treatment solution also
contained Ca, Mg, Fe, S, Mn, Zn, Cu, B, Co, and Mo,
and the pH was adjusted to 6.5 with 1N HCl or 1N NaOH.

 x Foliar N determined by micro-Kjeldahl.

 y Disease severity rated on a scale of 0 = no symptoms
to 6 = plant dead 30 days after inoculation with
Colletotrichum fragariae
isolate CF-63.

 z Mean separation by Least Significant Difference, P = 0.05.


Photosynthesis rate. The photosynthesis rate was similar on plants of all three clones and increased as treatment N levels increased (Table 1, Fig. 1). It was lowest on plants treated with the lower N concentrations (0 ppm N = 0.28 mg CO2/m/sec to 80 ppm N = 0.35 mg CO2/m/sec), increased on plants treated with 160 ppm N (0.47 mg CO2/m/sec), and nearly doubled on plants treated with 320 ppm N (0.87 mg CO2/m/sec). There was no significant effect on photosynthesis rate due to P and K levels or a significant interaction between N level and strawberry clone.


Nitrogen Source Studies

Two studies compared the effect of seven N sources each applied at three levels on anthracnose crown rot severity. In Nitrogen Source Study I, there were significant differences due to strawberry clone, N source, and N level, and there was a significant interaction between N source and N level (Table 4). ‘MSUS 98’ plants had lower disease severity ratings than ‘MSUS 37’ or ‘MSUS 70’ plants when averaged across all treatments (Table 5). Plants receiving 160 ppm N had higher disease severity ratings than plants receiving 0 ppm N or 40 ppm N. The highest disease severity ratings were from plants receiving 160 ppm of (NH4)2HPO4, (NH4)2SO4 and NH4Cl (Table 6). Plants receiving 160 ppm Ca(NO3)2 had disease severity ratings as low as the ratings of plants receiving no N; however, the N content of their leaves was as high as those in any other fertility treatment.


Table 4. Analysis of variance results of two studies comparing the effect of seven N treatments applied at three levels to greenhouse grown strawberry plants on anthracnose crown rot disease severity ratingsx.

Study Source DF F value Pr > F
N source I Clone 2 7.69               0.0006
N source 7 4.60               0.0001
Level 2 44.82               0.0001
Source × Level 5 6.05               0.0001
N source II Clone 2 32.03               0.0001
N source 7 4.15               0.0003
Level 2 5.26               0.006   
Source × Level 5 3.06               0.0002

 x Eight weeks after the initiation of treatment applications, plants were inoculated with a suspension of Colletotrichum fragariae conidia, incubated in a dew chamber at 30°C and 100% relative humidity for 48 hr, and then held in the greenhouse at 22 ± 7°C.


The results of Nitrogen Source Study II were similar to those of Nitrogen Source Study I (Table 4). Plants of ‘Tangi’ and ‘MSUS 74’ had higher disease severity ratings than plants of ‘MSUS 70’ and ‘MSUS 98’ (Table 5). Plants receiving no N had lower disease severity ratings than those receiving 40 ppm N or 120 ppm N. The lowest disease ratings due to N source were from plants receiving the Ca(NO3)2, KNO3, and NH4NO3 treatments


Table 5. Main treatment effects of two studies comparing the effects of N source and level on anthracnose crown rot disease severity ratings (DR)w of strawberry plants grown in sand culture in the greenhouse.

Study N source DRx N level DR
N source I (NH4)2SO4       4.3 ay 160 ppm 4.7 a
(NH4)2HPO4       4.3 a 40 ppm 2.7 b
KNO3       3.8 a 0 ppm 2.4 b
NH4Cl       3.7 ab Clone
NH4NO3       3.5 ab
NaNO3       3.4 ab MSUS 70 3.9 a
Ca(NO3)2       2.9 bc MSUS 37 3.7 a
None       2.4 c MSUS 98 2.9 b
Study N source DRz N level DR
N source II (NH4)2SO4       4.8 a 120 ppm 3.9 a
NaNO3       4.2 ab 40 ppm 3.6 a
(NH4)2HPO4       4.1 abc 0 ppm 2.6 b
NH4Cl       3.9 abc Clone
NH4NO3       3.6 bcd Tangi 5.0 a
KNO3       3.1 cd MSUS 74 4.7 a
Ca(NO3)2       2.8 d MSUS 70 2.7 b
None       2.6 d MSUS 98 2.1 b

 w Disease severity rated on a scale of 0 = no symptoms to 6 = plant dead 30 days after inoculation with Colletotrichum fragariae isolate CF-63.

 x Disease ratings (DR) in Nitrogen Study I are the average of five plants each of ‘MSUS 37,’ ‘MSUS 70,’ and ‘MSUS 98’ treated with three levels of N (0, 40, and 160 ppm N).

 y Mean separation within studies and within columns and factors by Least Significant Difference, P = 0.05.

 z Disease ratings (DR) in Nitrogen Study II are the average of four plants each of ‘Tangi,’ ‘MSUS37,’ ‘MSUS70,’ and ‘MSUS98’ treated with three levels of N (0, 40, and 120 ppm N).


Table 6. Effect of N source and level applied to strawberry plants grown in sand culture in the greenhouse in Nitrogen Source Study I on anthracnose crown rot disease ratings (DR) and percent foliar N (% FN). Each value is the average of five plants each of the strawberry clones MSUS 37, MSUS 70, and MSUS 98.

N Source Treatment N level Treatment N level
40 ppm 160 ppm 40 ppm 160 ppm
DRx % FNy
(NH4)2SO4      2.7 abcz       5.9 e       1.32 e      1.92 bc
(NH4)2HPO4      2.7 abc       5.8 e       1.33 e      1.89 bc
KNO3      2.9 bc       4.7 de       1.38 e      2.06 b
NH4Cl      1.4 a       5.9 e       1.26 e      2.10 ab
NH4NO3      3.2 bc       3.9 cd       1.32 e      1.60 cd
NaNO3      3.2 bc       3.7 bcd       1.31 e      1.96 b
Ca(NO3)2      2.9 bc       2.9 bc       1.49 de      2.30 a
No N      2.4 ab       1.01 f

 x Disease severity rated on a scale of 0 = no symptoms to 6 = plant dead 30 days after inoculation with Colletotrichum fragariae isolate CF-63.

 y Foliar N determined by micro-Kjeldahl.

 z Mean separation within factors by Least Significant Difference, P=0.05.


Implications for Control of Anthracnose Crown Rot on Strawberry

The source and level of N in fertilizers had a major effect on severity of anthracnose crown rot in strawberry, whereas the level of P and K in fertilizers did not. Plants that received N as Ca(NO3)2 had less severe anthracnose crown rot symptoms than plants that received N in the ammonium form. Even though these trials were conducted using greenhouse grown potted plants, the results should apply to strawberries grown in very sandy soils such as is typical in Florida.

These results suggest that strawberry growers can modify their fertilizer regimes by using Ca(NO3)2 as their N source to reduce anthracnose crown rot severity while maintaining the higher foliar N levels needed to achieve good growth and production. As expected foliar N increased in this study as treatment N levels increased, and there was no effect on foliar N levels due to P and K levels. In a similar study, the severity of anthracnose fruit rot (caused by C. acutatum) was reduced when Ca(NO3)2 was applied to greenhouse grown strawberries (11). Similar results were also reported for control of Fusarium diseases of tomato (15) and chrysanthemum (14). There was a significant difference in foliar N levels among the strawberry clones which suggests that N applications should be tailored to each cultivar; however, the trend of reduced anthracnose severity with nitrate fertilizer was similar on the five strawberry clones in these trials which indicates that other cultivars would respond in the same way.


Acknowledgment

I thank Wanda S. Elliott for technical assistance.


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