Baseline and differential sensitivity of Peronophythora litchii (lychee downy blight) to three carboxylic acid amide fungicides

Authors


*E-mail: mgzhou@njau.edu.cn

Abstract

Downy blight, caused by Peronophythora litchii, is an important disease of lychee (litchi) plants in China. The in vitro sensitivities of various asexual stages of P. litchii to the three carboxylic acid amide (CAA) fungicides dimethomorph, flumorph and pyrimorph were studied with four single-sporangium isolates. None of the three fungicides affected zoospore discharge from sporangia, but they strongly inhibited mycelial growth (mean EC50 values of 0·075, 0·258 and 0·115 mg L−1, respectively); sporangial production (mean EC50 values of 0·085, 0·315 and 0·150 mg L−1, respectively); germination of cystospores (mean EC50 values of 0·140, 0·150 and 0·645 mg L−1, respectively); and germination of sporangia (mean EC50 values of 0·203, 0·5 and 0·743 mg L−1, respectively). As mycelial growth was the most sensitive stage to dimethomorph and pyrimorph, it was chosen to test baseline sensitivities to the three fungicides. In 2007, from 131 isolates collected in Fujian, Guangdong and Guangxi provinces, 127, 116 and 113 isolates were used to establish baseline sensitivity for dimethomorph, flumorph and pyrimorph respectively. Isolates from different provinces exhibited similar baseline sensitivity to the same fungicide. Baseline sensitivities to dimethomorph, flumorph and pyrimorph were distributed as unimodal curves, with mean EC50 values of 0·082 (± 0·01), 0·282 (± 0·047), and 0·115 (± 0·032) mg L−1, respectively. This information will serve as a baseline for tracking future changes in sensitivities of P. litchii populations to these three CAA fungicides.

Introduction

Peronophythora litchii, a transitional species between Phytophthora and Peronospora (Chi et al., 1984; Voglmayr, 2003; Zhang et al., 2007), is the causal agent of lychee (litchi) downy blight (Fig. 1), one of the major diseases of lychee (Litchi chinensis) in Southern China. The pathogen attacks fruits, panicles and new shoots, causing panicle rot, withering and watery brown spots on fruits which later sporulate, producing downy white sporangiophores. The pathogen also causes significant post-harvest losses. It can over-winter via mycelium and oospores in the remnants of leaves. The pathogen is polycyclic in nature, resulting in the dispersal of inoculum over an extended time period and wide area during the season. Crop losses due to this disease become extensive under the conditions of hot, humid, rainy weather that often occurs in Southern China during the period when fruit develops. Chemical control is the primary method for controlling lychee downy blight from March to July each year.

Figure 1.

Lychee (Litchi chinensis) fruits, healthy (left) and affected by Peronophythora litchii (right).

Carboxylic acid amide (CAA) fungicides are used extensively worldwide against different downy mildews, such as Pseudoperonospora cubensis (Zhu et al., 2007b) and Plasmopara viticola and Phytophthora species such as P. infestans but they are not active against Pythium species. The mode of action of these chemicals has not yet been fully elucidated. There are proposals for inhibition of phospholipids biosynthesis (Griffiths et al., 2003) and for interference with cell wall deposition (Kuhn et al., 1991; Cohen & Baider, 1995; Dereviagina et al., 1999; Matheron & Porchas, 2000; Bagirova et al., 2001; Reuveni, 2003; Gisi & Sierotzki, 2008). This mode of action is different from other available fungicides such as strobilurins, phenylamides, dithiocarbamates active against oomycetes. The CAA fungicide dimethomorph was first introduced in China in 1996 by BASF. Flumorph, another CAA fungicide, was developed by Shenyang Research Institute of Chemical Industry, China in 1994, and its commercial use began in 1999. Recently another CAA fungicide, pyrimorph, was synthesized in 2007 by Gengyun Chemical Co., China. Both flumorph and pyrimorph can be classified into the same group with dimethomorph based on their chemical structures and activity against target pathogens (Liu et al., 1999; Chen et al., 2007) and cross-resistance activity (data not shown). To the authors’ knowledge, there is less information about interaction between P. litchii and CAA fungicides due to a narrow distribution of lychee in Southern China, Northern Vietnam, India, Malaysia and Australia (Mitra, 2002). Dimethomorph and flumorph have been used commercially on lychee for less than 10 years in China, and pyrimorph has not been used at all to date. In order to develop a sound recommendation for lychee downy blight management after the market introduction of CAA fungicides, information regarding the sensitivity distribution of P. litchii isolates to these fungicides is required.

Therefore, the objectives of the current study were: (i) to detect the sensitivities of P. litchii at different asexual stages to dimethomorph, flumorph and pyrimorph; (ii) to develop a simple method for measuring the sensitivity distribution of P. litchii isolates to dimethomorph; and (iii) to establish the baseline sensitivity distribution of P. litchii in China to all three compounds.

Materials and methods

Fungicides

Technical-grade dimethomorph (98·3% active ingredient (a.i.), BASF China Co., Ltd.), flumorph (92·5% a.i., Shenyang Research Institute of Chemical Industry), and pyrimorph (98% a.i., Gengyun Chemicals) were used in all assays. The fungicides were dissolved in methanol to provide stock solutions containing 1000 mg a.i. L−1 of dimethomorph, flumorph or pyrimorph. Serial dilutions were made with sterile distilled water as required, stored at 4°C in the dark to preserve fungicide activity, and were added to autoclaved media (lima bean agar (LBA) or water agar) when cooled to approximately 50°C. The volumes of methanol concentration were less than 0·25% of the testing solution. This concentration of methanol did not affect mycelial growth or the germination of encysted spores or sporangia of P. litchii (data not shown). The control always contained the same methanol concentration as the test samples in the experiments.

Origin and collection of isolates

During the epidemic season of lychee downy blight in 2007, isolates of P. litchii were collected from diseased fruit with typical symptoms from Guangxi, Guangdong and Fujian provinces, the major lychee fruit production regions in Southern China. To enhance sporulation, diseased fruit samples were washed with distilled water three times, placed in a moist chamber and incubated at 28°C for 24 h. Sporangia were transferred onto LBA plates and then incubated at 28°C for 8 h (LBA was prepared by boiling 60 g L−1 lima beans for 1 h, adding deionized H2O to a final volume of 1·0 L, and then 16 g L−1 agar powder). Pieces of agar containing single sporangia were removed and placed onto new LBA plates. Growing mycelium was transferred to LBA slants for storage at 25°C. A total of 131 single-sporangium isolates were collected from 15 different commercial lychee orchards, without a history of CAA fungicide application. Among the total isolates, 44 were collected from Guangxi province, 65 from Guangdong province, and 22 from Fujian province (Table 1). Each orchard was sampled once and c. 10 isolates were generated from each sampling. Different orchards in the same province were at least 40 km apart, and orchards in different provinces were at least 400 km apart (Fig. 2).

Table 1.  Origin of 131 isolates of Peronophythora litchii collected from diseased lychee fruit in 2007 for baseline sensitivity study
RegionFarm codeCultivarNo. isolates tested
GuangxiLingshanFeizixiao 4
YulinFeizixiao10
NanningLuomici 8
LiuzhouFeizixiao 9
QinzhouHeiye13
GuangdongDongwanGuiwei10
ZengchengGuiwei12
ConghuaLuomici14
GaozhouHeiye 9
MaomingBaila11
GenzizhengBaitangyin 9
FujianZhangzhouWuye 5
FuzhouWuye 4
MinghouYuanhong 6
PutianYuanhong 7
Figure 2.

Origin of Peronophythora litchii samples from orchards in Southern China. Five, six and four orchards sampled in Guangxi (left), Guangdong (middle) and Fujian (right) provinces, respectively.

Four isolates (Gd1, Ch76, Yn145 and L5) were chosen randomly from the isolate collection to compare the sensitivity of different asexual stages. Isolates Gd1 and Ch76 were from two different orchards in Guangdong province, while Yn145 and L5 were from two different orchards in Guangxi province.

Sensitivity of mycelial growth

Isolates were incubated on LBA plates at 25°C for 5 days. Individual agar discs (5-mm-diameter) were removed from the edge of an actively growing culture and placed face up on the centre of a Petri dish (9-cm-diameter) containing LBA to which the fungicides had been added at various concentrations. The final concentrations tested for dimethomorph were 0, 0·05, 0·08, 0·11, 0·14, 0·17 and 0·20 mg L−1; for flumorph 0, 0·0625, 0·125, 0·25, 0·5 and 1·0 mg L−1; and 0, 0·05, 0·10, 0·20, 0·40 and 0·80 mg L−1 for pyrimorph. Cultures were incubated at 25°C in the darkness for 6 days. For each fungicide, three replicates per concentration were used, and the experiment was performed twice. For each plate, the colony diameter was determined as the mean of two measurements taken perpendicular to each other; the initial diameter of the mycelial plug (5 mm) was subtracted.

Sensitivity of sporangial production

The sporangial production of P. litchii in the presence of different concentrations of the different fungicides was examined using a modification of a previously described method of sporangial quantification (Caten & Jinks, 1968). Six colonized agar plugs, 5 mm in diameter, were excised from each replicate plate from the in vitro mycelial growth sensitivity assay. Three continuous plugs were harvested at approximately 2 mm from the colony margin and another three from approximately 2 mm from the edge of the initial central inoculum plug. The six plugs were put into a single 2-mL reaction tube with 1·0 mL of sterile deionized H2O and agitated for 20 s with a laboratory vortex to dislodge the sporangia. Sporangia were quantified with a haemocytometer and the number of sporangia cm−2 was calculated. This experiment was conducted twice with three replicates.

Sensitivity of the direct germination of sporangia

Sporangia were harvested at approximately 5 mm distance from the colony margin as described above. Dimethomorph, flumorph and pyrimorph were added to LBA plates to achieve concentrations of 0, 0·0975, 0·195, 0·39, 0·78, 1·56 and 3·12 mg L−1. Sporangial suspensions (0·12 mL) containing 1 × 105 sporangia mL−1 were spread on the plates and incubated at 30°C in the dark for 12 h (Chi et al., 1984). Germination was quantified at three sites on each Petri dish by counting 100 sporangia per site. A sporangium was scored as germinated if the germ tube had reached at least the full length of the sporangium. The experiment was conducted twice, with three Petri dishes for each treatment.

Sensitivity of zoospore formation, discharge and cystospore germination

For testing the sensitivity of zoospore formation and discharge to the three CAA fungicides, sporangial suspensions containing 1 × 105 sporangia mL−1 were harvested as described earlier. Aqueous mixtures of dimethomorph, flumorph and pyrimorph at different concentrations were added to an equal volume (500 µL) of the sporangial suspension in a single 2-mL reaction tube to give final concentrations of 0, 6·25, 12·5, 25, 50, 100 and 200 mg L−1. Sporangial suspensions were incubated at 16°C in the dark for 2 h for zoospore formation and discharge. Released zoospores were quantified microscopically by counting three replicate fields of 100 sporangia each. A sporangium was scored as discharged if the sporangium was empty. Three replicates for each concentration of the fungicides were used and the experiment was conducted twice.

For testing the sensitivity of germination of encysted zoospores (cystospores), sporangial suspensions described above were centrifuged for 4 min, and maintained at 16°C in the dark for 30 min. Zoospore suspension was collected from the supernatant. Dimethomorph and flumorph were added to 1·6% w/v water agar plates after sterilization to achieve concentrations of 0, 0·05, 0·10, 0·15, 0·2, 0·25, 0·30 and 0·50 mg L−1; and for pyrimorph of 0, 0·16, 0·32, 0·63, 1·25, 2·5 and 5 mg L−1. Zoospore suspension (0·12 mL) containing 1 × 105 zoospores mL−1 was spread on the plates and incubated at 16°C in darkness for 4 h (Chi et al., 1984). Germination was quantified at three sites on the agar plates by counting 100 sporangia per site. A cystospore was scored as germinated if the germ tube had reached at least the length of the cystospore. The experiment was conducted twice, with three dishes for each treatment.

Determination of the baseline sensitivity of P. litchii to CAA fungicides

Baseline sensitivity of P. litchii was determined using the mycelial growth test for dimethomorph, flumorph and pyrimorph as described above.

Data analysis

Data from repeated experiments were combined for analysis, because variances between experiments were not statistically significant. The concentration of each fungicide causing 50% (EC50) and 90% (EC90) reduction in mycelial growth, sporulation, inhibition of zoospore discharge, and germination of cystospores and sporangia was estimated from the fitted regression line of the log-transformed percentage inhibition plotted against the log-transformed fungicide concentration (Brandt et al., 1988).

Results

Sensitivity of mycelial growth

All three chemicals had good activity against mycelial growth of the pathogen (Table 2). Sensitivity was greatest to dimethomorph, with average EC50 and EC90 values of 0·075 and 0·140 mg L−1, respectively, followed by pyrimorph, with average values of 0·115 and 0·525 mg L−1, respectively. Higher EC50 and EC90 values were recorded for flumorph, averaging 0·258 and 0·655 mg L−1, respectively. In the absence of a fungicide, the average mycelial growth diameter for P. litchii on LBA plates after 6 days at 25°C was 64 mm.

Table 2.  Sensitivities of different asexual stages of four isolates of Peronophythora litchii to dimethomorph, flumorph and pyrimorpha
 Mycelial growthSporangial productionCystospore germinationSporangial germinationZoospore discharge
EC50EC90EC50EC90EC50EC90EC50EC90EC50EC90
  • a

    EC50 and EC90 values (mg L−1) are the concentrations of each fungicide causing 50 and 90% reduction, respectively, in mycelial growth, sporangial production, cystospore germination, sporangial germination and zoospore discharge compared to fungicide-free control.

Dimethomorph
 Gd10·070·130·090·180·140·330·250·71> 200> 200
 Ch760·060·150·080·170·130·280·160·54> 200> 200
 Yn1450·080·160·080·170·140·340·140·59> 200> 200
 L50·090·120·090·180·150·340·260·69> 200> 200
Flumorph
 Gd10·150·520·250·760·150·430·561·96> 200> 200
 Ch760·250·560·320·810·140·440·502·09> 200> 200
 Yn1450·320·810·350·830·150·430·402·99> 200> 200
 L50·310·730·340·780·160·470·542·27> 200> 200
Pyrimorph
 Gd10·130·500·140·260·611·630·604·44> 200> 200
 Ch760·090·330·150·330·541·730·774·0> 200> 200
 Yn1450·090·960·150·350·681·540·684·06> 200> 200
 L50·150·310·160·400·751·840·929·64> 200> 200

Sensitivity of sporangial production

All three compounds were active against sporangial production with average EC50 and EC90 values of 0·085 and 0·175 mg L−1, respectively for dimethomorph; average values of 0·150 and 0·335 mg L−1, respectively for pyrimorph; and average values of 0·315 and 0·795 mg L−1, respectively for flumorph (Table 2). The average number of sporangia developing on LBA plates in the absence of a fungicide after 6 days inoculation at 25°C was more than 3 × 106 cm−2.

Sensitivity of the direct germination of sporangia

All three compounds were very active against direct germination of sporangia (Table 2). Highest sensitivity was noted for dimethomorph, with average EC50 and EC90 values of 0·203 and 0·633 mg L−1, respectively; followed by flumorph, with average values of 0·50 and 2·328 mg L−1, respectively; and pyrimorph, with average values of 0·743 and 5·535 mg L−1, respectively. The percentage germination of sporangia in the absence of fungicides after 12 h at 30°C was c. 80%.

Sensitivity of zoospore formation, discharge, and cystospore germination

None of the fungicides had an effect on zoospore formation and their discharge from sporangia, even with EC50 values higher than 200 mg L−1 for each fungicide, but cystospore germination was strongly inhibited (Table 2). Dimethomorph was most effective, with average EC50 and EC90 values of 0·140 and 0·323 mg L−1, respectively; followed by flumorph, with average values of 0·150 and 0·443 mg L−1, respectively; and pyrimorph, with average values of 0·645 and 1·685 mg L−1, respectively. In the absence of fungicides usually > 90% of cytospores germinated after 4 h incubation at 16°C.

Baseline sensitivity to dimethomorph, flumorph and pyrimorph

A total of 127, 116 and 113 single-sporangium isolates of P. litchii were tested for their sensitivity to dimethomorph, flumorph and pyrimorph using the mycelial growth assay. There was no evidence of geographical variation in the sensitivity of P. litchii to each fungicide (Table 3). For dimethomorph, the EC50 values for inhibition of mycelial growth ranged from 0·061 to 0·113 mg L−1 with a mean of 0·082 ± 0·010 mg L−1, representing a sensitivity range of 1·85 times; for flumorph, EC50 values ranged from 0·180 to 0·447 mg L−1 with a mean of 0·282 ± 0·047 mg L−1, representing 2·48 times; and for pyrimorph, EC50 values ranged from 0·060 to 0·222 mg L−1 with a mean of 0·115 ± 0·032 mg L−1, representing 3·70 times.

Table 3.  Baseline sensitivity of field isolates of Peronophythora litchii to dimethomorph, flumorph and pyrimorph
Province sampledDimethomorph EC50aFlumorph EC50Pyrimorph EC50
No.bRangeMeancSDNo.RangeMeanSDNo.RangeMeanSD
  • a

    Mean EC50 values (mg L−1) of baseline sensitivity of P. litchii to dimethomorph, flumorph and pyrimorph were calculated for inhibition of mycelial growth on LBA plates.

  • b

    Number of tested isolates.

  • c

    Mean values followed by the same letter within the same column were not significantly different using least significant difference tests at P = 0·05.

Guangxi440·061–0·1130·083a0·012310·180–0·4470·285a0·052290·060–0·2110·111a0·036
Guangdong610·061–0·1070·081a0·010650·184–0·4010·285a0·050640·071–0·2150·118a0·030
Fujian220·064–0·0930·081a0·008200·221–0·3440·268a0·027200·075–0·2320·112a0·037

The frequency distribution of the EC50 values for all isolates to dimethomorph, flumorph and pyrimorph were all unimodal curves (Fig. 3). There were six, seven and six points on the distribution curve for sensitivity to dimethomorph, flumorph, and pyrimorph, respectively. There was no resistant subpopulation among the isolates used in the study for each fungicide.

Figure 3.

Distributions of baseline sensitivity to dimethomorph, flumorph and pyrimorph in isolates of Peronophythora litchii from Southern China determined by inhibition of mycelial growth in vitro. Isolates were sampled from orchards never exposed to CAA fungicides. A total of 127, 116 and 113 isolates of P. litchii were investigated for sensitivity to dimethomorph, flumorph and pyrimorph, respectively.

Discussion

Neither of the CAA fungicides had an effect on zoospore formation or discharge from sporangia, but all three strongly inhibited mycelial growth, sporulation, and germination of cystospores and sporangia of P. litchii in vitro. These findings are similar to observations generated with other CAA fungicides, such as benthiavalicarb (Reuveni, 2003) and iprovalicarb (Dutzmann, 1999) with other pathogens. The in vitro sensitivities of P. litchii at various stages to dimethomorph, flumorph and pyrimorph were seen to differ slightly. Both dimethomorph and pyrimorph were active against mycelial growth, while the most sensitive stage to flumorph was the germination of cystospores. Moreover, of the three CAA fungicides, dimethomorph exhibited the strongest inhibitory effects on all asexual stages of P. litchii based on EC50 and EC90 values. This is supported by recent field trials where dimethomorph exhibited better field performance than the other two fungicides (Wang et al., 2006).

These data and also previous studies with other peronosporomycetes have shown that CAA fungicides are strong inhibitors of sporangial and cystospore germination (Cohen & Baider, 1995; Cohen & Gisi, 2007; Zhu et al., 2007a). However, these stages are influenced by different factors such as maturity of sporangia or nutrition (Kao & Leu, 1980; Chi et al., 1984). Since the mycelial growth assay was sensitive to all CAA fungicides and this method is reliable, less time-consuming and less labour-intensive compared with the other methods described, the mycelial growth test was the method of choice for the assessment of the baseline sensitivity with numerous isolates of P. litchii. The baseline distributions of P. litchii were all unimodal and described a narrow range of the EC50 values for dimethomorph, flumorph, and pyrimorph, and were similar to the sensitivity range of Phytophthora infestans to dimethomorph (Yuan et al., 2005) and flumorph (Yuan et al., 2006) and of Phytophthora capsici to dimethomorph (Anthony, 2007).

The number of orchards and the number of isolates per orchard were large enough to reflect the population in the sampled regions. Therefore, these results can be used in the future to monitor potential sensitivity changes to these CAA fungicides in populations of P. litchii.

Acknowledgements

This study was funded by BASF Company and the Scientific and Technological Support Program from the Chinese Government (2006BAD08A03). The authors thank the three companies for providing technical grade fungicide.

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