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Crop Protection 30 (2011) 1e9

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Crop Protection

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Rice spikelet rot disease in China e 1. Characterization of fungi associatedwith the disease

Shi-Wen Huang a, Ling Wang a, Lian-Meng Liu a, Shao-Qing Tang a, De-Feng Zhu a, Serge Savary b,*

aChina National Rice Research Institute (CNRRI), Hangzhou, Zhejiang 310006, PR Chinab International Rice Research Institute (IRRI), Plant breeding, Genetics and Biotechnology Division, DAPO Box 7777, Metro Manila, Philippines

a r t i c l e i n f o

Article history:Received 25 October 2009Received in revised form6 July 2010Accepted 10 July 2010

Keywords:Rice spikelet rot diseasePathogenBiological characteristicsSystematicsMolecular characterization

* Corresponding author.E-mail address: s.savary@cgiar.org (S. Savary).

0261-2194/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.cropro.2010.07.010

a b s t r a c t

Rice spikelet rot disease (SRD) is an emerging disease of rice panicle in China, which affects both riceyield and grain quality. Four fungal pathogens were isolated from diseased rice grains. Morphologicalobservation, biological testing and molecular characterization led to identify these fungi as Fusariumproliferatum, Bipolaris australiensis, Curvularia lunata and Alternaria tenuis. The four fungi can grow from10 �C to 40 �C, and from pH 5 to pH 10. The most suitable temperature range is 25 �Ce30 �C, however theoptimal pH for sporulation of these fungi varies greatly. The four fungi can grow on media supplementedwith different carbon and nitrogen sources. These differences in carbon and nitrogen requirementssuggest differences in trophism, and have large effects on hyphal growth and spore production. Theresults suggest that rice SRD is caused by various fungi with diverse physiological characteristics.

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1. Introduction

Panicle diseases of rice, have generally received comparativelylittle attention. False smut (Ustlilaginoidea virens), kernel smut(Tilletia barclayana), scab (Fusarium graminearum, other Fusariumspecies being also considered as saprophytes) have long beenconsidered minor rice diseases that do not warrant specific controlmeasures. The prevention of grain discoloration (associated witha wide range of organisms) has been generally associated to betterpre- and post-harvest measures; while many fungi found onglumes and panicles have commonly been seen as organisms thatonly are found onweakened plants or moribund tissues (Ou, 1987).Although Ou’s view still essentially holds true today, the wideadoption of rice plant types with different panicle morphology incropping systems partly due to their photoperiod-insensitivity andhave high yield potential has led to a significant change in theimpact of such rice diseases in rice production.

Spikelet rot disease (SRD), for instance, has become an emergingrice disease in China, as losses caused by the disease have beenregularly increasing over recent years. SRD appears to be associatedwith a number of factors, including the popularization of large-panicle, non-glutinous plant type found in both modern inbredvarieties and hybrids. Our understanding of the causal agents,

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epidemiological factors and the levels of susceptibility of cultivatedrice varieties and hybrids to of this disease is limited. Such lack ofknowledge hampers the development of suitable disease riskforecasts and control measures.

This article reports the efforts conducted at the China NationalRice Research Institute (CNRRI) during the recent years. Here, wereport the results of isolation, molecular, morphological, andmicrobiological characterization from samples collected duringsurveys on micro-organisms associated with typical SRD symp-toms, and discuss the implications of SRD as a new rice diseasecomplex. The importance of and possible options for managementof the disease are addressed in a companion article.

2. Materials and methods

2.1. Disease samples

Disease samples with typical SRD symptoms were collected atthe early dough stage from infected panicles of cvs. Nipponbare andXiushui 09 (inbred varieties) and of hybrid rice Yongyou 8060grown in field plots at the Experimental Research Station of CNRRI,Hangzhou, Zhejiang.

2.2. Standard culture media for isolations

Unless stated otherwise, all isolations were performed usingPotato Dextrose Agar (PDA; potato 200.0 g, dextrose 20.0 g, agar

S.-W. Huang et al. / Crop Protection 30 (2011) 1e92

20.0 g, and distilled water 1 L) or Czapek’s Agar medium (sucrose30.0 g, sodium nitrate 2.0 g, magnesium sulfate 0.5 g, ferric sulfate0.01 g, dipotassium hydrogen phosphate 1.0 g, potassium chloride0.5 g, agar 13.0 g and distilled water 1 L).

2.3. Isolation and purification of pathogen isolates

Diseased grains were first washed with distilled sterile waterand isolations were performed according to a standard blockmethod for pathogenic fungus isolation (Fang, 1996). The observedcolonies were then examined under the microscope. Individualspores were picked for single-spore cultures on PDA. Alternately,individual pieces of hyphaewere sub-cultured on PDA. Four distinctfungal isolates, SF1, SF2, SF3 and SF4, were obtained.

2.4. Morphological characteristics of cultured isolates

Plugs from PDA cultures were sub-cultured at 28 �C. The appear-ance of seven day-old colonies was recorded and their morphologicalcharacteristics in cultureswerequantified.Cultureswere scrapedwith100 ml of water 0.05% (v/v) solution of Tween 20 (polysorbate-20;

Fig. 1. a. Conidia of Fusarium proliferatum, b. Systemic position of F. prolifer

Shanghai Commonalty Pharmacy Ltd. Company), and spore suspen-sions for microscopic examination were prepared.

2.5. Molecular identification of fungal species

ThetotalDNAof the isolateswasextracted frommycelia followingLee and Taylor (1990)with somemodifications. The rDNA of internaltranscribed spacer regions including the 5.8S gene was amplifiedusing a pair of primers, ITS1 (50-TCCGTAGGTGAACCTGCGG-30) andITS4 (50-TCCTCCGCTTATTGATATGC-30) (White et al., 1990). A 50 mlvolume of PCR reactionmixture containing 5 ml 10� PCR buffer, 4 mldNTPs (2.5 mM each), 1.25 ml each of primers ITS1 and ITS4 (100 mmeach), 0.25 ml Taq polymerase (5 U mL�1), 1 ml (1 ng ml�1) DNA and37.25 ml sterile distilled water was prepared for each sample. Theamplificationwas performedwith aDNA thermal cyclerMP (TaKaRa,Ltd.). The cycle parameters were an initial denaturation at 94 �C for2 min, followed by 30 cycles of denaturation at 94 �C for 50s,annealing at 55 �C for 1 min, extension at 72 �C for 1 min, and finalextension at 72 �C for 10 min. The amplified products were purifiedusing the Agarose Gel Purification Kit (TaKaRa, Ltd.) according to themanufacturer’s instruction. The PCR products were then ligated to

atum and its homologous genera constructed based on ITS sequences.

S.-W. Huang et al. / Crop Protection 30 (2011) 1e9 3

the pMD18 vector (TaKaRa, Ltd.) at 4 �C overnight according to theinstructions provided by supplier. The ligated plasmidswere used totransform DH5a competent cells, which were then plated on LBmedium containing 100 mg/L ampicillin, isopropyl-b-D-thio-galactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl-b-D-gal-actopyranoside (X-gal). Plasmid DNA was isolated from whitecolonies and sequenced using the BigDye terminator v3.1 cyclesequencing kit (Applied Biosystems).

The sequencing results were analyzed using BLAST (http://www.ncbi.nlm.nih.gov/). Testing and comparison of similarity of ITS genesequence of our isolates and sequences deposited in Genbank wereperformed.

Based on the proportion of different nucleotide sites, DNAsequence similarity was calculated using the DNAMAN softwarepackage (version 4.0, Lynnon Biosoft, Vaudreuil, Quebec, Canada).Neighbor-joining tree showing thephylogenetic relationships among

Fig. 2. a. Conidia of Bipolaris australiensis, b. Systemic position of B. austral

the four isolates (SF1, SF2, SF3 and SF4) and other organisms whoseITS sequences are deposited in GenBank were constructed (Whiteet al., 1990). Distances in the ITS-5.8S rDNA region were determinedusing Kimura’s two-parameter model (Kimura, 1980). Bootstrappingwas performed using 1000 re-samples of the sequence.

2.6. Influence of culture condition on the hyphal growth andsporulation

The influence of temperature on hyphal growth and sporulationwas assessed from subcultures and growth measurements from5 mm diameter colonized plugs on PDA in Petri dishes at 5, 10, 15,20, 25, 30, 35 and 40 �C, with three replications per isolate. Colonydiameters were measured along two orthogonal directions afterfive days. Each colony was scraped with 100 ml of Tween 20 watersolution (0.05%, v/v) to determine number of spores.

iensis and its homologous genera constructed based on ITS sequences.

S.-W. Huang et al. / Crop Protection 30 (2011) 1e94

The influence of pH on hyphal growth and sporulation wasquantified using the samemethods as above. Six pH levels (5, 6, 7, 8,9 and 10) were tested by adding NaOH and HCl to adjust the pHvalue of the PDA medium.

The influence of carbon and nitrogen sources on hyphal growthand sporulation was analyzed using Czapek’s Agar medium. Forthe carbon source tests, 30.0 g of six different sources of carbonwere used: dextrose, fructose, starch, maltose, lactose and xylose.These were substituted to the sucrose in the original Czapekculture medium. For the nitrogen source tests, six different sour-ces were tested: proline, histidine, asparagine, ammonia sulfate,carbamide and sodium nitrite. These were substituted to sodiumnitrate in the Czapek culture medium. A 5 mm colony plug fromeach of the four isolates was placed in the middle of the Petridishes. These experiments were repeated 3 times. Determinationof colony diameter and sporulation were made as describedabove.

Analyses of variance and Duncan’s new multiple range methodcomparisons were conducted on all data of the above experimentsusing the Data Processing System (DPS; Tang and Feng, 2007).

Fig. 3. a. Conidia of Curvularia lunata, b. Systemic position of C. lunata

3. Result and analysis

3.1. Isolation, purification and identification of pathogens

Four fungal isolates with different morphological features wereobtained and purified from diseased rice grain samples and labeledas SF1, SF2, SF3 and SF4.

3.1.1. Identification of isolate SF1On PDA, isolate SF1 was white at early stage and then became

pink, with abundant aerial mycelium and flocculence. It generatedtwo types of conidia, large or small. Large conidiawere few, fusiformor sickle-shaped, slightly bending, with 3e10 septa, long apical celland clear odospora at the base, 17.5e47.5 � 3.5e5.5 mm. Smallconidiophores were colorless and produced rapidly, tapering at oneend and obtuse at the other end; with obvious ground cytoplasmroot, 2-4 septa and with a size of 16.5e26.4 � 3.3e4.9 mm (Fig. 1a).

The ITS sequence of SF1 was entered in GenBank with theaccession number (AN) FJ040179. This isolate had 100% similaritywith the ITS gene sequence of Fusarium proliferatum (accession

and its homologous genera constructed based on ITS sequences.

S.-W. Huang et al. / Crop Protection 30 (2011) 1e9 5

number is FPU34574). Similarity coefficients with F. acutatum (AN:AY213653) and F. annulatum (AN: AY213654) were 99.46% and99.64%, respectively. Considering the amount, shape and attachmentmode of the large and small conidia as well as the growth rate andcolor of the colony, together with evidence from ITS nucleotidesequence comparison, this isolate was identified as F. proliferatum(Deuteromycotina/Hyphomycetes/Tuberculariales/Tuber-culariaceae/Fusarium) (Fig. 1b).

3.1.2. Identification of isolate SF2On PDA, colonies of isolate SF2 were fuzzy, beige, with a central

apophysis, tighten with trim edges. The color of hyphae was hazel,

Fig. 4. a. Conidia of Alternaria tenuis, b. Systemic position of A. tenuis

smooth, septate, always branched, of 1.5e3.5mmwide. The conidiumpeduncle was brown, septate, straight or bended, basal cell wasincrassate and semi-spherical, having various branches and alwaysfascicled. The tip of conidia was lateral, straight, fusiform or oblongoval, narrowand thin at the twoends, normally 3 false septum, sandybeige to brown, with the size as 22.5e36.5 � 8.0e12.0 mm, theumbilical point was inside the basal cell (Fig. 2a).

The ITS sequence of SF2 was entered in GenBank with theaccession number FJ040180. The similarity of the ITS sequence ofSF2 with that of Cochliobolus australiensis (AN: AY923860) andBipolaris spicifera (AN: AY253918) are 99.82% and 99.29%, respec-tively. Moreover, it was found to belong to the same branch as

and its homologous genera constructed based on ITS sequences.

a a

a

a

a a

b

c d a a

b

a

a

a a

a

a

a a

ab

b

c

a a

b

b

a a

ab b

b

a a

b

c

Co l

o ny

di am

et er

( mm

)

Te mp er at ur e (˚ C)

Fig. 5. Variation of five day-old colony diameters with temperature of four fungal species associated with rice spikelet rot disease.

S.-W. Huang et al. / Crop Protection 30 (2011) 1e96

C. australiensis and B. spicifera. Together with the conidia shape andthe position of cleft hilum, the data led to identifying SF2 asC. australiensis (imperfect stage: Bipolaris australiensis) (Deuter-omycotina/Hyphomycetes/Hyphomycetales/Dematiaceae/Bipolaris) (Fig. 2b).

3.1.3. Identification of strain SF3On PDAmedium, the colonies of SF3were brown, circular, flat and

short lanose. Thehyphaewerecolorlessorhazel, smooth, septate,withmany branches, 1.7e4.0 mmwide. Conidium peduncle was solitary orfascicled, septate, with few branches, straight or bent, genuflected attheir top, brown of a lighter color at their top. Conidia were solitary,three-septate, with a top adnation, bent, oval or near-fusiform, darkerin color than the conidiumpeduncle,18.0e32.0� 8.5e15.5mmin size,with no apparent cleft hilum, (Fig. 3a).

The ITS sequence of SF3 was entered in GenBank with theaccession number FJ040177. Homology comparison between rDNAITS sequence of isolate SF3 with those already in GenBank showedthat SF3 has high homologieswith species in Cochliobolus, Curvulariaand Bipolaris, with the highest homology, 100% with Cochlioboluslunatus (AN: EF189917). This homology, togetherwithmorphologicalcharacteristics led to identifying SF3 as C. lunata (imperfect stage:Curvularia lunata) (Deuteromycotina/Hyphomycetes/Hyphomyce-tales/Dematiaceae/Curvularia) (Fig. 3b).

3.1.4. Identification of isolate SF4Colonies of SF4 on PDA were flat, flocculent, beige on the surface

andbrownat theback.Hyphaewerehazel, smooth, septate, branched,

Table 1Effect of temperature on in vitro sporulation of four fungal species associated with spike

Temperature(�C)

Fusarium proliferatum Bipolaris australiensis

�106 spores ml�1 % of optimalsporulation

�106 spores ml�1 % of optsporula

4 5.25 � 0.66dA 0.31 1.00 � 0.58dB 0.5010 6.00 � 0.76dA 0.35 1.75 � 0.16dB 0.8815 150.00 � 11.55cA 8.82 9.00 � 0.50dB 4.5020 600.00 � 39.30bA 35.29 75.00 � 7.64bcB 37.5025 1700.00 � 57.74aA 100.00 55.00 � 12.58cC 27.5028 1700.00 � 57.74aA 100.00 85.00 � 2.89bC 42.5030 625.00 � 8.66bA 36.76 200.00 � 16.07aC 100.0035 6.25 � 1.09dC 0.37 1.00 � 0.58dC 0.5040 0.00 � 0.00dA 0.00 0.50 � 0.50dA 0.25

Notes: Measurements were made after eight days of culture. Small letters refer to compadifferent letters indicating significant difference at P ¼ 0.05 using Duncan’s multiple ran

2.0e3.5 mmwide. Conidia peduncles were solitary or fascicled, erect,brown, and septated. Conidia (22.0e58.0 � 10.0e17.5 mm) were soli-tary or concatenate, with a reverse club-or pear-shaped, hazel tobrown, smooth, with 1e9 horizontal septa, 1 to 6 vertical or inclinedsepta, with slight constrictionswhere transversal septawere formed.The basal cell (1.0e55.0 � 1.0e6.5 mm) showed a beak, whereas thetopmost cell (22.0e58.0 � 10.0e17.5 mm) was coniform or columni-form (Fig. 4a).

The ITS sequence of SF4 was entered in GenBank with theaccession number FJ040178. Homology comparison showed that,among the 100 fungi whose sequences were available in GenBankand close to SF4, 76 were in the genus Alternaria. A classificationtree constructed with the sequences of Alternaria species showedthat A. tenuis was the closest to SF4 (Provide the homology here).These results, combined with morphological characteristics, led toidentifying SF4 as A. tenuis (Deuteromycotina/Hyphomycetes/Hyphomycetales/Dematiaceae/Alternaria) (Fig. 4b).

3.2. Influence of different temperatures on each isolate

As shown in Fig. 5, each of the four isolate could grow between10 �C and 40 �C; however, the most rapid growth for all isolateswas between 25 �C and 30 �C. B. australiensis nevertheless stillgrew rapidly at 40 �C; while C. lunata and A tenuis were nottolerate temperatures below 15 �C.

Sporulation of all isolates was favored by temperaturesranging from 25 to 30 �C (Table 1). The most suitable temperaturefor the sporulation of F. proliferatum was 25e28 �C (1.7 � 109

let rot disease.

Curvularia lunata Alternaria tenuis

imaltion

�106 spores ml�1 % of optimalsporulation

�106 spores ml�1 % of optimalsporulation

0.04 � 0.00fB 0.01 0.00 � 0.00dB 0.000.09 � 0.01fC 0.02 0.00 � 0.00dC 0.004.75 � 0.14fB 1.19 18.75 � 1.13dB 4.6942.50 � 1.32eB 10.63 13.50 � 0.76dB 3.3885.00 � 7.64dC 21.25 250.00 � 25.17bB 62.50250.00 � 5.77bD 62.50 400.00 � 26.46aB 100.00400.00 � 0.00aB 100.00 236.25 � 15.53bC 59.06112.50 � 10.10cA 28.13 87.50 � 6.61cB 21.880.25 � 0.14fA 0.06 0.50 � 0.29dA 0.13

rison of values along columns and letters in capitals refer to comparison along lines,ge test.

Table 2Effect of different pH values on sporulation of four fungal species associated with spikelet rot disease.

pH value Fusarium proliferatum Bipolaris australiensis Curvularia lunata Alternaria tenuis

5 7400.00 � 57.73aA 165.00 � 2.89dC 465.00 � 5.77dB 95.00 � 23.63bcC6 4000.00 � 152.75bcA 425.00 � 13.22cB 450.00 � 0.00cB 120.00 � 5.77bC7 3300.00 � 57.74dA 575.00 � 14.43bB 550.00 � 26.45bB 170.00 � 10.00aC8 3700.00 � 152.75cA 270.00 � 30.55dBC 525.00 � 37.74bcB 75.00 � 8.66cdC9 4100.00 � 152.75bA 800.00 � 76.37aB 650.00 � 28.86aB 90.00 � 15.27bcC10 2700.00 � 28.87eA 450.00 � 28.86cC 650.00 � 28.86aB 41.00 � 0.58dD

Notes: Values indicate millions spores per milliliter. Measurements were made after eight days of culture. Small letters refer to comparison of values along columns and lettersin capitals refer to comparison along lines, different letters indicating significant difference at P ¼ 0.05 using Duncan’s multiple range test.

Fig. 6. Variation of five day-old colony diameters with culture medium pH for four fungal species associated with rice spikelet rot disease.

S.-W. Huang et al. / Crop Protection 30 (2011) 1e9 7

spores per ml), and the sporulation of this fungus was stronglyreduced when temperature was below 10 �C or above 35 �C. Themost suitable sporulation temperature for B. australiensis was30 �C (2 � 108 spores per ml), and sporulation was suppressed bytemperatures below 15 �C or above 35 �C. For C. lunata, thehighest sporulation was observed at 30 �C (4 � 108 spores perml), and its sporulation was suppressed by temperatures below15 �C or above 40 �C. The most suitable temperature for thesporulation of A. tenuis was 28 �C (4 � 108 spores per ml), andsporulation of the fungus was suppressed when temperatureswere below 10 �C and above 40 �C.

3.3. Effect of pH of culture medium on the growth and sporulationof the isolates

All of the four isolates were able to grow between pH 5 to pH 10.F. proliferatum however appeared to have a narrower range (poorer

Fig. 7. Variation of five day-old colony diameters with carbon sources

growth at pH¼ 5), while the ranges of pHwere quite similar for theother three fungi (Fig. 6).

The four isolates differred significantly in their optimum pH forsporulation. The sporulation of F. proliferatum, B. australiensis,C. lunata, and F. proliferatum were the highest at pH 5 (7.4 � 109spores per ml), pH 9 (8.0 � 108 spores per ml), pH 9 to pH 10(6.5 � 108 spores per ml) and pH 7 (1.7 � 108 spores per ml),respectively (Table 2).

3.4. Influences of different carbon sources on fungal growth andsporulation of the isolates

All four isolates were able to grow on different types of carbonsources (Fig. 7). Sucrose, maltose and lactose were the commonbest carbon sources for the four species, but species differred intheir utilization of the seven sources of tested carbon. F. pro-liferatum was able to use all kinds of carbon sources very well.

for four fungal species associated with rice spikelet rot disease.

Table 3Influence of different carbon sources on sporulation of four fungal species associated with spikelet rot disease (106 spores/g of carbon source).

Carbonsource

Fusarium proliferatum Bipolaris australiensis Curvularia lunata Alternaria tenuis

�106 spores ml�1 % of optimalsporulation

�106 spores ml�1 % of optimalsporulation

�106 spores ml�1 % of optimalsporulation

�106 spores ml�1 % of optimalsporulation

Dextrose 5900.00 � 104.08aA 196.67 135.00 � 12.58aB 0.90 155.00 � 14.43aB 5.17 50.00 � 4.08dB 1.67Fructose 3675.00 � 72.17cA 122.50 30.00 � 5.00dB 0.20 17.00 � 0.58eB 0.57 115.00 � 6.12aB 3.83Cane sugar 1525.00 � 43.30fA 50.83 80.00 � 5.00bB 0.53 65.00 � 2.89dB 2.17 75.00 � 2.04cB 2.50Starch 1825.00 � 87.80eA 60.83 50.00 � 5.77cB 0.33 90.00 � 5.77cB 3.00 95.00 � 2.04bB 3.17Maltose 2050.00 � 111.50deA 68.33 90.00 � 0.00bB 0.60 90.00 � 5.77cB 3.00 16.00 � 0.00fB 0.53Lactose 2200.00 � 115.47dA 73.33 35.00 � 2.89cdB 0.23 130.00 � 5.77bB 4.33 58.00 � 4.32dB 1.93Xylose 4400.00 � �115.47bA 146.67 25.00 � �2.89 dB 0.08 80.00 � �5.77cdB 2.67 35.00 � �1.87eB 1.17

Notes: Measurements were made after eight days of culture. Small letters refer to comparison of values along columns and letters in capitals refer to comparison along lines,different letters indicating significant difference at P ¼ 0.05 using Duncan’s multiple range test.

S.-W. Huang et al. / Crop Protection 30 (2011) 1e98

B. australiensis used starch better than the other three species asa carbon source, but utilized xylose poorly. C. lunata and A. tenuiscould not use xylose very well, but their utilization of the othersources of carbon was similar.

Different carbon sources showed strong effects on the sporula-tion of the four fungal isolates (Table 3). Of the seven sources ofcarbon, dextrose was the best for the sporulation of F. proliferatum,B. australiensis, and C. Lunata, whereas that of A. tenuiswasmaximalwith fructose. The sporulation of B. australiensis was the least offour isolates in all seven sources of carbon.

3.5. Influences of different nitrogen sources on fungal growth andsporulation

The type of nitrogen source had a large influence on the growthof four isolates (Fig. 8). Proline, histidine, asparagine and carbamidefavored most hyphal growth of all species, followed by sodiumnitrite and sodium nitrate, while ammonia sulfate was very poorlyutilized. Growth of F. proliferatumwas the fastest with proline as thenitrogen source; of B. australiensis the fastest with asparagine; of C.lunata the fastest with histidine and carbamide; and of A. tenuis thefastest with histidine, asparagine and carbamide.

Different nitrogen sources tested also showed strong effects onthe sporulation of each species (Table 4). The use of ammoniasulfate nitrogen strongly reduced the sporulation of all species, andinhibited the sporulation of B. australiensis. The maximal sporula-tion levels of F. proliferatum, B. australiensis, C. lunata and A. tenuiswere achieved with histidine and asparagines, asparagine andsodium nitrate, histidine, and sodium nitrate, respectively, asnitrogen sources.

Fig. 8. Variation of five day-old colony diameters with nitrogen source

4. Discussion

Rice grain may be infected by various organisms before or afterharvest, causing discoloration. A large number of fungi and bacteriaare associated with the discoloration of rice grain (Ou, 1987). Twomain groups of organisms can be considered associated with ricegrain discoloration. The first group consists of fungi that are moreor less pathogenic and infect the grains before harvest. The secondgroup includes storage moulds, which usually are saprophytes anddevelop after harvest (Ou, 1987). The first group includes C. miya-beanus (Drechslera oryzae), Pyricularia oryzae, A. padwickii, Giberellafujikuroi, G. zeae, Phoma sorghina, Helicoceras oryzae and otherspecies in genera Nigrospora, Epicoccum, Curvularia, Alternaria, andHelminthosporium and Drechslera (Ou, 1987).

Tullis (1936) isolated many fungi from discoloured grain in theUSA. The most commonwere Alternaria spp. and C. lunata, followedby A. padwickii and phoma spp., more rarely D. oryzae and Fusariumspp. There were many unidentifiable isolates.

The initial symptomof SRD is a reddishdiscoloration atfloweringand milk stage, and which turns to brown and black at the ripeningand fully mature stage. SRD not only causes grain discoloration butalso grainmalformation. Four fungiwere isolated fromSRDdiseasedgrain. The present study indicates that the pathogens involved inrice SRD are quite different. SRD is partly but not completely similarto the commonly, and rather diverse, rice discolorations that havebeen reported in the literature for many years (see Ou, 1987). Asidefrom the fungal pathogens it is associated with, SRD is a very recentdisease in some of the most productive rice-producing provinces ofChina; its symptoms are quitepeculiar, especiallywith respect to thefact that entire panicles can be infected, and entire fields can be

s for four fungal species associated with rice spikelet rot disease.

Table 4Influence of different nitrogen sources on sporulation of four fungal species associated with spikelet rot disease.

Nitrogen source Fusarium proliferatum Bipolaris australiensis Curvularia lunata Alternaria tenuis

�106 spores ml�1 % of maximalsporulation

�106 spores ml�1 % of maximalsporulation

�106 spores ml�1 % of maximalsporulation

�106 spores ml�1 % of maximalsporulation

Proline 600.00 � 28.87aA 85.71 8.00 � 0.71dB 20.00 13.00 � 1.53dB 8.97 40.00 � 5.00bB 53.33Histidine 700.00 � 50.00aA 100.00 24.00 � 2.16bC 60.00 145.00 � 8.66aB 100.00 16.00 � 1.00dC 21.33Asparagine 700.00 � 57.74aA 100.00 40.00 � 1.08aB 100.00 55.00 � 8.66cB 37.93 11.00 � 0.58dB 14.67Ammonia sulfate 1.00 � 0.00cB 0.14 0.00 � 0.00eB 0.00 0.50 � 0.29dB 0.34 3.60 � 0.95eA 4.80Carbamide 600.00 � 50.00aA 85.71 15.00 � 1.08cB 37.50 15.00 � 1.53dB 10.34 40.00 � 0.00bB 53.33Sodium nitrate 70.00 � 2.89cB 10.00 40.00 � 2.04aC 100.00 90.00 � 7.64bA 62.07 75.00 � 2.52aB 100.00Sodium nitrite 460.00 � 5.77bA 65.71 15.00 � 0.71 cC 37.50 14.00 � 1.00 dC 9.66 29.00 � 2.08 cB 38.67

Notes: Measurements were made after eight days of culture. Small letters refer to comparison of values along columns and letters in capitals refer to comparison along lines,different letters indicating significant difference at P ¼ 0.05, using Duncan’s multiple range test.

S.-W. Huang et al. / Crop Protection 30 (2011) 1e9 9

diseased. At present, no study has addressed the occurrence,epidemical regularity and the management of SRD. The infectionprocess and (multiple-pathogen) disease cycle are unclear andrequire specific research. Preliminary results (Huang et al., 2011)suggest that the main transmission route of rice SRD is that thedifferent pathogens invade floral organs and glumes at the riceheading and flowering stage. The disease is favored by windy andrainy conditions. The pathogens then exploit the rice grain nutrientsto reproduce quickly, leading to symptoms.

Our results indicated that pathogens involved in SRD greatlydiffer in their morphological and cultural characteristics andresponses to temperature and pH ranges. These pathogens onlyinvade the glume shells and grains, and have not been isolated inother plant tissues. Molecular biology tools helped identifying thefour isolated species as F. proliferatum, B. australiensis, C. lunata andA. tenuis. This provides a basis for further research on this diseasecomplex.

Different rice varieties and hybrids seem to show strongdifference in susceptibility to SRD (Huang et al., 2011). Generally,closed-panicle plant types, such as round-grained non-glutinousrice inbreds and hybrids, seem to be more easily and severelyinfected. Growing a single susceptible variety over large areas alsoseems to favor the SRD. In addition, some pathogens that cause SRDcan produce mycotoxin. F. proliferatum produces fumonisin (Tariqet al., 1995; Javed et al., 1993; Stankovic et al., 2007; Abbas et al.,1999), while A. tenuis produces tenuazonic acid, alternariol, alter-toxin-I and alternariol monomethyl ether (Logrieco et al., 1990;Panigrahi and Dallin, 1994). These toxins are harmful to animalsand humans. Therefore, further study on the pathogenic fungi andon the measures to effectively control SRD are needed.

Acknowledgements

This study was partly supported by the Special Fund for Agri-cultural Industry (nyhyzx07-049), Special Funds from the CentralCommittee of the Commonweal Scientific Research Institute(100015), funding from the Scientific and Research Support Plan onAgriculture (2006BAD08A04), the Three-Agriculture Five-Party

Scientific Cooperation Project of the Zhejiang Province (SN200711).We wish to thank the Editors for their assistance in publishing thiswork.

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