Plant Pathology (2013) 62, 49–58 Doi: 10.1111/j.1365-3059.2012.02613.x

Population genetic evidence that basidiospores play animportant role in the disease cycle of rice-infectingpopulations of Rhizoctonia solani AG-1 IA in Iran

F. Padasht-Dehkaeia†, P. C. Ceresinibc*†, M. Zalab, S. M. Okhovvata,

M. J. Nikkhaha and B. A. McDonaldb

aDepartment of Plant Protection, College of Agriculture and Natural Resources, University of Tehran, Plant Protection Building,

Faculty Street, 31587-11167 Karaj, Iran; bPlant Pathology, Institute of Integrative Biology, ETH Zurich, Universitaetstr. 2, 8092 Zurich,

Switzerland; and cDepartamento de Fitossanidade Engenharia Rural e Solos, UNESP - Universidade Estadual Paulista, Campus de

Ilha Solteira, 15385-000 Ilha Solteira, Sao Paulo, Brazil

The fungus Rhizoctonia solani AG-1 IA causes sheath blight, one of the most important rice diseases worldwide. The first

objective of this study was to analyse the genetic structure of R. solani AG-1 IA populations from three locations in the Ira-

nian Caspian Sea rice agroecosystem. Three population samples of R. solani AG-1 IA isolates were obtained in 2006 from

infected rice fields separated by 126–263 km. Each field was sampled twice during the season: at the early booting stage and

45 days later at the early mature grain stage. The genetic structure of these three populations was analysed using nine micro-

satellite loci. While the population genetic structure from Tonekabon and Amol indicated high gene flow, they were both

differentiated from Rasht. The high gene flow between Tonekabon and Amol was probably due mainly to human-mediated

movement of infested seeds. The second objective was to determine the importance of recombination. All three populations

exhibited a mixed reproductive mode, including both sexual and asexual reproduction. No inbreeding was detected, suggest-

ing that the pathogen is random mating. The third objective was to determine if genetic structure within a field changes over

the course of a growing season. A decrease in the proportion of admixed genotypes from the early to the late season was

detected. There was also a significant (P = 0Æ002) increase in the proportion of loci under Hardy–Weinberg equilibrium.

These two lines of evidence support the hypothesis that basidiospores can be a source of secondary inoculum.

Keywords: Gene flow, genetic structure, mixed reproductive mode, recombination, Thanatephorus cucumeris

Introduction

Sheath blight caused by the multinucleate fungus Rhizoc-tonia solani anastomosis group AG-1 IA (teleomorphThanatephorus cucumeris) is a major rice disease world-wide (Savary et al., 2000; Willocquet et al., 2000; Zhang,2007) with crop losses ranging from slight to 50% (Cuet al., 1996; Slaton et al., 2003). Sheath blight is the mostimportant component of the rice sheath disease complex,which includes two other diseases: sheath spot, caused bymultinucleate R. oryzae (teleomorph Waitea circinata),and aggregate sheath spot, caused by binucleate R. ory-zae-sativae (teleomorph Ceratobasidium oryzae-sativae)(Gunnell & Webster, 1984; Johanson et al., 1998;Lanoiselet et al., 2007). Worldwide, rice sheath spot and

*E-mail: paulo.ceresini@bio.feis.unesp.br†These authors contributed equally to this manuscript.

Published online 26 March 2012

ª 2012 The Authors

Plant Pathology ª 2012 BSPP

aggregate sheath spot are either considered as minordiseases or gaining importance as emerging diseases(Lanoiselet et al., 2007).

The rice sheath blight pathogen R. solani AG-1 IA wasfirst described in Japan at the beginning of the 20th cen-tury (Kozaka, 1975) and has since been reported in mostrice-growing regions (Ou, 1985). In Iran, rice has beencropped since 400 BC but the first report of rice sheathblight was in Mazandaran province in 1981 (Torabi &Binesh, 1984). Following the introduction of susceptibledwarf cultivars and the use of higher rates of nitrogen fer-tilizer, rice sheath blight became the second most impor-tant fungal disease in northern Iran (Okhovvat, 1999),exceeded only by rice blast (Mousanezhad et al., 2010).

Despite the importance of this rice pathogen, the popu-lation biology of the fungus in Iran remains poorlyunderstood. As reported in other rice-producing areas ofthe world (Lee & Rush, 1983; Kobayashi et al., 1997),R. solani AG-1 IA in Iran has been considered an asexualfungus on rice because the asexual reproductivestructures have been most commonly observed, includingsclerotia, mycelia from rice debris in the field and myceliafrom infested seeds (Binesh & Torabi, 1985; Okhovvat,

49

50 F. Padasht-Dehkaei et al.

1999). In most countries where the sexual cycle has beenobserved, basidiospores are not thought to contributesignificantly to the epidemiology of rice sheath blight(Kozaka, 1975). Hymenia producing basidiospores havebeen observed in Texas (USA) on infected rice as well ason alternative hosts, including soybean, sorghum andmaize (Jones & Belmar, 1989). The occurrence of hyme-nia and basidiospores from T. cucumeris was alsoreported recently in Mazandaran province, Iran (Khosraviet al., 2011). Basidiospores are formed midseason duringthe booting stage on rice (Hashiba & Kobayashi, 1996).

The paradigm of strictly asexual reproduction in rice-infecting and also in soybean-infecting populations ofR. solani AG-1 IA from Asia (China and India), NorthAmerica (Texas and Louisiana, USA) and Latin America(Brazil and Venezuela) has been challenged by populationgenetic studies using RFLP and microsatellite markers(Rosewich et al., 1999; Linde et al., 2005; Bernardes deAssis et al., 2008, 2009; Gonzalez-Vera et al., 2010).These studies indicated a mixed reproductive scenario inwhich the pathogen undergoes cycles of sexual recombi-nation in addition to clonal propagation, suggesting thatbasidiospores may play a significant role in sheath blightepidemiology.

The disease biology and dispersal ecology of the rice-infecting R. solani AG-1 IA has been extensively studied.The rice sheath blight disease cycle usually starts fromoverwintered asexual primary inoculum, mainly fungalmycelia or buoyant sclerotia present in the soil, irrigationwater or stubble (Savary et al., 1997). The primary inocu-lum initiates infection in the lower canopy of newly trans-planted rice plants (Hashiba & Kobayashi, 1996). Ingeneral, the secondary inoculum is attributed to mycelialstrands, growing from the primary lesions as runninghyphae on the surface of leaves and sheaths to establishnew secondary lesions in the upper parts of the canopy(Savary et al., 1997).

Rice-infecting R. solani AG-1 IA survives as myceliumand sclerotia in soil. Recurring disease cycles increaseinoculum in the soil (Ogoshi, 1987). The pathogen alsosurvives on seeds (Ogoshi, 1987). In fact, seedborne inoc-ulum is known to play a significant role in overwinteringand long distance dispersal of the sheath blight pathogen.In Iran, the percentage of infested seeds in the improvedrice cultivar Amol2 harvested from naturally infestedfields varied from 22 to 39% in Mazandaran province(Binesh & Torabi, 1985). Eight months after harvesting,the fungal inoculum was still viable on the seeds (10–19% of seeds infested). After sowing these infested seedsin sterilized soil, disease incidence on rice seedlings variedfrom 6 to 12% (Binesh & Torabi, 1985). Further evidencesupporting the survival of R. solani AG-1 IA in seeds andthe role of seedborne inoculum in the transmission of thepathogen from seed to seedlings in the field was foundin Uttarakhand, India (Sivalingam et al., 2006). Thetransmission from seed to seedlings was 6Æ2%, similar tothat reported earlier in Iran (Binesh & Torabi, 1985).

Little is known about the genetic structure of R. solaniAG-1 IA populations in Iran. In a study of 216 isolates

sampled from the three most important rice-growingprovinces (Guilan, Golestan and Mazandaran), 16groups were identified using soluble protein patterns inSDS-PAGE and 38 groups were found using thin layerchromatography (TLC) of sclerotial lipids (Tajickghan-bari et al., 2002). Genetic diversity was also characterizedusing rDNA markers (ITS-5Æ8S), which differentiated twogroupsamong 102 isolates (Tajickghanbari et al., 2005).

Knowledge of the population genetic structure ofR. solani AG-1 IA in Iran is needed to understand theevolutionary processes that have shaped these popula-tions in the local agroecosystem and their implicationsfor the effectiveness of disease management strategies(Milgroom & Fry, 1997; McDonald & Linde, 2002;Milgroom & Peever, 2003). For example, analysis of thedistribution of gene diversity within and among popula-tions of the pathogen can be used to detect patterns ofpathogen migration (Beerli & Felsenstein, 2001). A highdegree of similarity between geographically distant popu-lations provides evidence for long distance gene flowbetween them, probably associated with inoculum dis-persal via infected seeds. If seedborne inoculum plays asignificant role in R. solani AG-1 IA dispersal, thenimproved seed testing and seed treatment may berequired to reduce the spread of sheath blight. Thus thefirst objective was to determine the genetic structure ofR. solani AG-1 IA populations from three locations in theIranian Caspian Sea rice agroecosystem. Based on earlierfindings of significant regional gene flow (Bernardes deAssis et al., 2009; Gonzalez-Vera et al., 2010), the nullhypothesis was no population subdivision. Analysis ofpopulation genetic structure can also be used to assess therelative contributions of asexual and sexual reproductionin pathogen populations (Milgroom, 1996; Zhan et al.,1998). Hence, the second objective was to determine theimportance of recombination in Iranian populations ofR. solani AG-1 IA. Based on earlier findings (Rosewichet al., 1999; Bernardes de Assis et al., 2008, 2009; Ciampiet al., 2008; Gonzalez-Vera et al., 2010), the null hypoth-esis was that populations would be random mating withlocal dispersal of a few clones. An alternative hypothesiswas that geographically distinct populations would havedifferent degrees of clonality. The third objective was todetermine if genetic structure within a field changes overthe course of a growing season. The null hypothesis wasthat there would be no change in structure between earlyand late season populations of R. solani AG-1 IA. Thealternative hypothesis was that genetic structure wouldchange as a result of differing contributions of sexual andasexual inoculum to the epidemic. If the disease cyclestarts from overwintered asexual inoculum, i.e. sclerotiapresent in the soil or on old straw, and then spreadsthrough mycelial growth or floating sclerotia during theearly phase of the epidemic, a high degree of clonalityearly in the season and maintenance of that clonality overtime would be expected. If basidiospores contribute sig-nificant inoculum to infection during the rice-growingseason, clonality should decrease and the populationsshould become more recombined and diverse over time.

Plant Pathology (2013) 62, 49–58

Population genetics of R. solani AG-1 IA from Iran 51

Control measures aimed at reducing basidiospore infec-tions, including fungicide applications during the forma-tion of the sexual phase, would reduce the amount ofsexual recombination and potentially reduce geneticdiversity in the corresponding pathogen populations.

Materials and methods

Fungal populations

Isolates of R. solani AG-1 IA were sampled in 2006 fromone naturally infested rice field in three different counties:Rasht (Guilan province), Tonekabon and Amol counties(Mazandaran province) along the Caspian Sea coast innorthern Iran. The field from Tonekabon was equidistantfrom Rasht (126 km apart) in the west and Amol(137 km apart) in the east (Fig. 1). This region includesmost of the rice production areas in Iran, representingapproximately 75% of total production (ASID, 1998). Ineach rice field, isolates were sampled twice during the sea-son: the first sample was collected approximately 1 weekafter disease was first observed between the end of the ricevegetative growth stage and the early booting stage(IRRI, 2002); the second sample was collected 45 dayslater, at the early mature grain stage, about 1 week beforeharvest. Diseased rice sheaths were collected in a grid pat-tern from 50 locations per field using five rows spaced10 m apart and locations separated by 10 m along eachrow. Thus each sample was 10 m distant from its nearestneighbour. To ensure that different sampling times repre-

35

36

37

38

39° North

0 50 100 km

Tonek

Rasht

48 50

Cas

Iran

Lon

Lat

itu

de

Direction of migration

To AmolTo Rasht

To Tonekabon

Migrants/generati

01·0

2·0 3·04

Migrat

Figure 1 Estimates of demographic parameters from the divergence amo

based on variation at nine microsatellite loci. Migration between populatio

program MIGRATE v. 2.3 (Beerli & Felsenstein, 2001). The directions of migr

arrows. Estimates of migration rates are with 95% confidence intervals.

Plant Pathology (2013) 62, 49–58

sented independent and temporally separated cycles ofinfection, only distinct lesions from upper rice sheathswere collected at the second time point. The followingrice cultivars were cropped in each location: Shafagh andFajr (in Amol), Deylemani (in Tonekabon) and Khazar(in Rasht). All four rice cultivars are considered suscepti-ble to sheath blight, with no difference in disease resis-tance rating (Okhovvat, 1999). Infected sheaths were putinto paper envelopes and dried at room temperature for2–4 days, then stored at )20�C. The fungus was isolatedfrom diseased tissue on water agar (WA) amended with50 mg L)1 each of streptomycin sulphate and penicillinG (Stevens-Johnk & Jones, 1994).

Germinated hyphal tips were transferred onto potatodextrose agar (PDA) and saved as pure cultures. Only oneisolate was kept from each point in every field to avoidrepeatedly sampling clones. To obtain sclerotia for long-term storage of isolates, cultures were incubated for 10 to14 days at 27�C, then sclerotia were air-dried on a sterilebench and transferred to 1Æ5 mL tubes and stored at)20�C. Fungal mycelia production and DNA extractionwere done as described earlier (Linde et al., 2005). Theanastomosis group of each isolate was determined byselective amplification of the 28S ribosomal DNA(rDNA) using the specific primers designed for R. solaniAG-1 IA: R. solani AG-common primer (forward) 5¢-CTCAAACAGGCATGCTC-3¢ and R. solani AG 1- IAspecific primer (reverse) 5¢-CAGCAATAGTTGGTGGA-3¢ (Matsumoto, 2002). From a total of 300 collected iso-lates, the PCR assay detected 48 isolates that were not

abon

Tehran

52 54° East

pian Sea

gitude

on

Migrants/generation

·0

Amol

ion rate [M = θ*(m/μ)]01·0

2·0 3·04·0

ng rice-infecting populations of Rhizoctonia solani AG-1 IA from Iran

ns was estimated using an isolation with migration model using the

ation between recipient and donor populations are indicated by

52 F. Padasht-Dehkaei et al.

R. solani AG-1 IA. Thus only 252 isolates were includedin the population genetics analysis. All 252 isolates pro-duced sasakii-type sclerotia on PDA, typical of R. solaniAG-1 IA (Yang et al., 1989). Sequence analysis of the ITS-5Æ8S region of the ribosomal DNA indicated that the 48remaining isolates were R. oryzae-sativae, the pathogenassociated with the aggregate sheath spot disease on rice.

SSR amplification and fragment analysis

Nine microsatellite (SSR) loci were used to genotype the252 isolates of R. solani AG-1 IA using fluorescent-labelled primers (Zala et al., 2008). PCR was done sepa-rately for each SSR locus in a final volume of 20 lL of 10·PCR buffer (New England Biolabs), 0Æ1 mM of eachdNTP and 0Æ5 lM of each labelled (Applied Biosystems)and non-labelled (Microsynth) primer. The PCR was in aBiometra T-gradient thermocycler, programmed for thefollowing steps: 2Æ5 min at 96�C for initial denaturation,then 35 cycles of 96�C for 30 s, 50�C for 30 s, 72�C for30 s, and a final extension step of 72�C for 5 min. Theannealing temperature for locus TC10 was increased to55�C. The PCR amplicons were separated on an auto-mated sequencer (ABI 3730 DNA Analyzer, Applied Bio-systems) using Genescan 500 LIZ as size standard (Zalaet al., 2008). All nine loci were run in two distinct sets; set1: TC01, TC02, TC03, TC05 and TC06; set 2: TC07,TC10, TC12 and TC17. Locus TC17 is identical to TC13(Bernardes de Assis et al., 2008). Alleles were differenti-ated using GENEMAPPER v. 4 (Applied Biosystems). TwoR. solani AG-1 IA control isolates (TcO5VNZ-A1A1(Gonzalez-Vera et al., 2010) and TcO5CHN-Bej-1-13(Bernardes de Assis et al., 2009), both from rice) wereincluded in every run of 94 samples to test the consistencyof allele scoring for every SSR marker across runs andacross different studies.

Data analysis

In all analyses it was assumed that R. solani AG-1 IA is afunctional diploid, i.e. it is a dikaryon. All of the datawere consistent with this assumption. The range ofrepeats for each locus, the number of alleles, the numberof private alleles (i.e. those present in only one popula-tion), and allele frequencies were calculated using CON-

VERT v. 1.31 (http://www.agriculture.purdue.edu/fnr/html/faculty/rhodes/students%20and%20staff/glaubitz/software.htm).

Genotype diversity

A microsatellite multilocus genotype for each strain wasdetermined using GENODIVE v. 2.0b7 (http://www.bentley-drummer.nl/software/software/GenoDive.html). Isolatesthat had the same multilocus SSR genotype were treatedas clones. Several indices of clonal diversity were calcu-lated using GENODIVE, including: (i) the number of geno-types per population; (ii) site specific genotypes; (iii) theclonal fraction (or the proportion of fungal isolates origi-

nating from asexual reproduction); and (iv) Stoddart andTaylor’s genotypic diversity Go (Stoddart & Taylor,1988), scaled by the maximum number of expected geno-types (Grunwald et al., 2003). Bootstrapping was used totest whether pairs of populations differed in their geno-type diversity using 1000 permutations with subsamplingto match the size of the smallest population (Grunwaldet al., 2003).

Gene diversity and population differentiation

Nei’s unbiased gene diversity was estimated asn ⁄ (n)1) · (1)Ripi

2), where p is the observed fre-quency of the ith allele (Nei, 1978). Allelic richnesswas estimated as the mean number of alleles per locus(El Mousadik & Petit, 1996) for a standardized mini-mum clone-corrected sample size of 15 (using rarefac-tion, implemented in FSTAT v. 2.9.3.2, http://www2.unil.ch/popgen/softwares/fstat.htm). FSTAT was used totest whether groups of samples differed for allelic rich-ness and gene diversity. P-values for the significance ofthe pairwise comparisons were obtained after 1000permutations. F statistics were used to quantify differ-entiation between pairs of populations and to assessthe degree of population subdivision. Genetic distanceswere computed based on the number of allele differ-ences between two microsatellite haplotypes (Weir &Cockerham, 1984). The null distribution of pairwise Fstatistics under the hypothesis of no differentiationbetween two populations was obtained by permutatinghaplotypes between populations. Significance of thefixation indexes was tested using 1023 permutationsby a non-parametric approach using the program ARLE-

QUIN v. 3.11 (http://cmpg.unibe.ch/software/arlequin3/).

Hardy–Weinberg and gametic equilibrium tests

Associations within and among loci for each populationwere assessed using tests for Hardy–Weinberg equilib-rium (HWE) and multilocus association, respectively. AnHWE test analogous to Fisher’s exact test was used.P-values were obtained using a Markov chain MonteCarlo (MCMC) approach implemented in ARLEQUIN,generating an exact probability distribution not biased byrare alleles. The inbreeding coefficient (FIS) across lociwas also calculated to test for a significant deficit orexcess of heterozygotes based on 1000 permutations andimplemented in ARLEQUIN. Associations among loci weretested using Fisher’s exact test based on an MCMCalgorithm with 1000 batches and 1000 iterations ⁄ batch.This test was implemented by GENEPOP v. 3.4 (http://genepop.curtin.edu.au/), with correction for rare alleles.A Bonferroni correction was used to avoid false rejectionsof the null hypothesis due to the large number ofcomparisons performed. Two loci were considered ingametic equilibrium when their associated P-valuewas > 0Æ05 ⁄ n, where n is the number of comparisons.The index of association (IA) was also measured usingMULTILOCUS v. 1.3 (http://www.bio.ic.ac.uk/evolve/

Plant Pathology (2013) 62, 49–58

Population genetics of R. solani AG-1 IA from Iran 53

software/multilocus/index.html), with 1000 randomiza-tions and fixing missing data during randomizations.

Test for admixture or hidden population structure

Departures from HWE observed in some populationsof R. solani AG-1 IA could be caused by a Wahlundeffect (i.e. population admixture; Hartl & Clark,1997). To determine whether any individuals in a sam-ple were immigrants with respect to their referencegeographical population, a Bayesian statistical modelwas used implemented by STRUCTURE v. 3.03 (http://pritch.bsd.uchicago.edu/structure.html). This programcalculates the membership coefficients to each of thepopulations (Q) of every multilocus genotype sampled(which were assigned a priori to their reference popula-tions). Ten runs were performed with a burn-in periodof 10 000 iterations and MCMC simulations of100 000 iterations for each run.

Demographic parameters and historical migration

The effective population sizes and the historical migra-tion rates among the three populations were estimatedusing a maximum likelihood test based on the MCMCmethod implemented in MIGRATE v. 2.3 (http://popgen.sc.fsu.edu/Migrate/Migrate-n.html). Initial esti-mates of gene flow among populations were obtainedwith MIGRATE v. 2.3 using five replicates of 10 initial

Table 1 Measures of genetic diversity for rice-infecting Rhizoctonia solani AG-1 IA

Geographical

populations

(province and

county of origin) Growth stagea

Sample

size (n)bNumber of

genotypes

Site sp

genot

Guilan

Rasht county Early booting 48 24 13 (11

Early mature grain 48 15 4 (11

Mazandaran

Amol county Early booting 38 18 10 (8

Early mature grain 45 27 21 (6

Tonekabon county Early booting 40 18 9 (9

Early mature grain 33 21 13 (8

Overall Early booting 126 60 32 (28

Early mature grain 126 63 38 (25

Total 252 123

aAccording to the standard evaluation system for rice (IRRI, 2002).bSample size of each population.cNumber of genotypes shared with other populations are shown in brackedStoddart and Taylor’s genotypic diversity (Stoddart & Taylor, 1988).eSmall letters compare populations; means followed by the same letter ar

test for differences in clonal diversity indices between populations calcula

match the size of the smallest population.fNei’s unbiased gene diversity (Nei, 1978), also known as expected heterog

FSTAT v. 2.9.3.2 used to test whether pairwise samples differed for Nei’s u

permutations.hCalculated according to El Mousadik & Petit, 1996.

Plant Pathology (2013) 62, 49–58

chains and five long final chains and a static heatingscheme with four temperatures (1Æ0, 1Æ3, 2Æ6 and 3Æ9).

Results

Gene and genotypic diversity

A total of fifty alleles were detected for the nine SSR lociamong the 252 isolates of R. solani AG-1 IA. A range of3–13 alleles per locus (average of 5Æ6 alleles) existedacross the six populations. Gene diversities per locus andacross populations ranged from 0Æ11 to 0Æ73. Privatealleles were found in the populations from Amol (SSR lociTC01, TC06 and TC10), Rasht (TC06) and Tonekabon(TC12). Clonal fractions ranged from 0Æ36 to 0Æ69 withan average of 0Æ52 in the early booting stage and 0Æ50 inthe early mature grain stage (Table 1). The mean geno-typic diversity decreased from 13Æ1 to 7Æ6 in Rasht whilein Tonekabon it increased from 6Æ1 to 14Æ5 between thefirst and second collections (Table 1). A total of 93 dis-tinct multilocus genotypes were found, with 28 of thesegenotypes shared among populations. Genotypes werealso shared between growth stages in all three field popu-lations, making up 22 to 60% of the isolates in each field.All the genotypes from all six populations were heterozy-gous for at least one locus, except for genotype 75 (isolateTc08TR_291). The expected heterozygosity (HE = Nei’sunbiased gene diversity) varied from 0Æ37 to 0Æ53 acrosspopulations (Table 1). The population from Amol (sam-

from Iran

ecific

ypesc

Shared

genotypes

between

sampling

stages

Clonal

fraction

Stoddart’s

genotypic

diversity (Go)d,e HEf,g

Allelic

richnessg,h

) 10 0Æ50 13Æ1a 0Æ53a 3Æ78ab

) 10 0Æ69 7Æ6b 0Æ52a 3Æ44b

) 6 0Æ53 6Æ8b 0Æ37b 3Æ07c

) 6 0Æ40 9Æ2ab 0Æ46ab 3Æ66ab

) 7 0Æ55 6Æ1b 0Æ42ab 3Æ66ab

) 7 0Æ36 14Æ5a 0Æ48a 3Æ95a

) 23 0Æ52 8Æ7 0Æ44 3Æ50

) 23 0Æ50 10Æ4 0Æ49 3Æ68

ts.

e not significantly different (P = 0Æ05) based on pairwise bootstrap

ted with GENODIVE v. 2.0b7; 1000 permutations with subsampling to

zygosity, averaged over all loci, corrected for sample size.

nbiased gene diversity and allelic richness, based on 1000

54 F. Padasht-Dehkaei et al.

pled at the booting stage) had both significantly lower HE

(0Æ37) and lower allelic richness (3Æ07) than the popula-tion from Rasht at the same stage, while the populationfrom Tonekabon (sampled at early mature grain stage)had higher allelic richness (3Æ95) than the populationfrom Rasht. There was not a consistent pattern of varia-tion in HE across population pairs from different growthstages.

Population differentiation

Pairwise population differentiation was measured usingF-statistics. The overall FST value was 0Æ028 (P £ 0Æ001),indicating low differentiation among the three geographi-cal populations (Table 2). The population from Rasht (inGuilan province) was significantly differentiated fromTonekabon and Amol (in Mazandaran) for both growthstages; FST ranged from 0Æ032 (P £ 0Æ01) to 0Æ118(P £ 0Æ001). There was no differentiation between popu-lations sampled in the same field at two points in time(FST ranged from )0Æ016 to 0Æ021, P = NS) for any of thethree locations.

Hardy–Weinberg and gametic equilibrium tests

Results of Hardy–Weinberg equilibrium tests are shownin Table 3. All populations were in HWE for most of theloci (from 63 to 89% of the loci). There was an increase ofone locus in HWE between the early and late collectionsin all three populations (t-statistic = 25, d.f. = 2,P £ 0Æ002). The FIS values were not significantly differentfrom zero for any of the populations, consistent with anabsence of inbreeding. The tests for gametic disequilib-rium (GD) showed significant indices of multilocus asso-ciation IA (P £ 0Æ001; Table 3) for all populations.However, the proportion of locus pairs in significant dis-equilibrium varied temporally and across populations.While the populations from Tonekabon had the lowestproportion of locus pairs in disequilibrium (�6%) at bothtime points, 42% of the locus pairs from Rasht showedGD at the first collection while only 8% showed GD at

Table 2 Pairwise measures of genetic differentiation among rice-infecting popula

stages in northern Iran based on FST values

Region Population Growth stage

FSTa

Rasht county

Early

booting

Early

grain

Guilan Rasht Early booting –

Early mature grain )0Æ016NS –

Mazandaran Amol Early booting 0Æ097*** 0Æ118

Early mature grain 0Æ032** 0Æ040

Tonekabon Early booting 0Æ058*** 0Æ073

Early mature grain 0Æ041** 0Æ058

aGenetic distances were computed based on the number of different allelNSnot significant at P £ 0Æ05, ***P £ 0Æ001 and **P £ 0Æ01, based on 1023 p

the second collection. In Amol, GD of locus pairsincreased from 7% to 18% over time.

Test for admixture

A test was conducted for admixture implemented by theprogram STRUCTURE, and identified 11 likely admixedgenotypes across the three populations at the bootinggrowth stage (Table 3, Fig. 2). These genotypes repre-sented an average of 19% admixture. Within each popu-lation, admixture ranged from 17% (Amol andTonekabon) to 21% (Rasht). Admixture dropped signifi-cantly to 4% (t-statistic = 10Æ4, d.f. = 2, P = 0Æ009) atthe early mature grain stage in all three fields. Only twoadmixed genotypes were detected in the late-season col-lection: one in Rasht and one in Tonekabon.

Demographic parameters and historical migration

The population parameter theta (h) was used as a relativemeasure of effective population size. There was no signifi-cant difference in population size among the three popu-lations (h ranged from 0Æ95 to 1Æ07). Estimates ofdirectional gene flow indicated symmetrical historicalmigration among the three populations, with an averageof 2Æ1 migrants per generation (95% C.I. = 1Æ2 to 3Æ6;Fig. 1).

Discussion

A population genetic analysis was conducted of the ricesheath blight pathogen R. solani AG-1 IA from north-ern Iran. The null hypothesis was that the three fieldpopulations along the Caspian Sea would not be differ-entiated as a result of significant gene flow in thisregion. An alternative hypothesis was isolation-by-dis-tance. While the population genetic structure fromTonekabon and Amol (both from Mazandaran prov-ince) was consistent with high levels of gene flow, theywere both differentiated from the Rasht population(from Guilan province). Because each Iranian province

tions of Rhizoctonia solani AG-1 IA from rice fields sampled at two growth

Amol Tonekabon

mature Early

booting

Early mature

grain

Early

booting

Early mature

grain

*** –** 0Æ021NS –** )0Æ001NS 0Æ014NS –** 0Æ008NS 0Æ010NS )0Æ011NS –

es between two microsatellite haplotypes (Weir & Cockerham, 1984).

ermutations.

Plant Pathology (2013) 62, 49–58

Table 3 Tests for random association of alleles within each locus and between pairs of loci in rice-infecting populations of Rhizoctonia solani AG-1 IA from

Iran

Region Population Growth stage

Clone

corrected n

Admixed

genotypesa

Loci under HWEb

FISc

Gametic equilibrium test

Number % Number % IAd P-valuee

Locus pairs at

significant

disequilibriumf %

Guilan Rasht Early booting 24 5 20Æ8 6 ⁄ 9 66Æ7 )0Æ037 1Æ61 0Æ001 15 ⁄ 36 41Æ7

Early mature grain 15 1 6Æ7 7 ⁄ 9 77Æ8 0Æ025 1Æ10 0Æ001 3 ⁄ 36 8Æ3

Mazandaran Amol Early booting 18 3 16Æ7 5 ⁄ 8* 62Æ5 )0Æ053 1Æ03 0Æ001 2 ⁄ 28* 7Æ1

Early mature grain 27 0 0Æ0 6 ⁄ 8* 75Æ0 0Æ076 1Æ15 0Æ001 5 ⁄ 28 17Æ9

Tonekabon Early booting 18 3 16Æ7 7 ⁄ 9 77Æ8 )0Æ029 1Æ14 0Æ001 2 ⁄ 36 5Æ6

Early mature grain 21 1 4Æ8 8 ⁄ 9 88Æ9 0Æ111 1Æ05 0Æ001 2 ⁄ 36 5Æ6

Overallg Early booting 18Æ1A 6 69Æ0B )0Æ038 1Æ26 18Æ1

Early mature grain 3Æ8B 7 80Æ6A 0Æ074 1Æ10 10Æ6

P-value for

two-tailed t-statistic

0Æ009 0Æ002 0Æ46 0Æ60

aAdmixture was determined using an assignment test implemented by STRUCTURE v. 3.03.bHWE test was performed using an exact test analogous to the Fisher exact test, using a Markov chain with forecasted length of 100 000

implemented in ARLEQUIN v. 3.11; *one locus was monomorphic.cPopulation specific FIS indices were calculated using 1000 permutations with FSTAT v. 2.9.3.2.dIA is an index of multilocus gametic equilibrium (Agapow & Burt, 2001).eTesting H0 = complete panmixia based on 1000 randomizations; for diploid data, the two alleles at a locus are shuffled together

(associations between alleles at locus are maintained in the randomized data sets, thus the test is purely for associations

between loci; Agapow & Burt, 2001).fNumber of locus pairs with significant disequilibrium according to Fisher exact test (probability test) using both a Markov chain

with 1000 batches and 1000 iteration ⁄ batch, implemented by GENEPOP v. 3.4, after Bonferroni correction for multiple comparisons.gA and B indicate that the overall means of admixed genotypes and of loci under HWE were significantly different at P £ 0Æ009 and P £ 0Æ002,

respectively, according to the t-statistic.

Population genetics of R. solani AG-1 IA from Iran 55

has its own set of recommended rice cultivars, exchangeof rice seeds is common among farmers within aprovince but limited between provinces (ASID, 1998). Itis hypothesized that the high gene flow betweenTonekabon and Amol was due mainly to human-mediated within-province movement of infested seedswhile the limited gene flow between populations fromMazandaran and Guilan provinces reflects restrictedseed exchange. Although currently subdivided, thepopulations from the two Iranian provinces haveexchanged migrants historically (Fig. 1), indicating thattheobservedsubdivisionmighthavearisenonlyrecently.

Despite the relatively low levels of seed transmissiondescribed in the literature (ranging from 6% to 12%;Binesh & Torabi, 1985; Sivalingam et al., 2006), thesetransmission rates are probably sufficient to contribute tothe increase in fungal inoculum where the disease isalready present and certainly could explain the spread ofthe pathogen to other locations. These findings suggestthat the seed testing and treatment measures already inplace in Guilan and Mazandaran provinces should bestrengthened. In particular, seed treatment with eradicantfungicides would be warranted to prevent long distancedispersal among rice growing areas in Iran. Rice seedtreatment is done by the Iranian Agricultural SupportServices Company (ASSC), but it is unusual among farm-ers (ASID, 1998). In addition to exchanging infected seed,farmers often trade rice straw as animal feed and share

Plant Pathology (2013) 62, 49–58

harvesting equipment (Torabi & Binesh, 1984; Binesh &Torabi, 1985), thus there are many mechanisms in placeto promote pathogen dispersal in this area.

The expectations for sexually recombining heterothal-lic fungi are high genotypic diversity, low clonal fraction,neutral markers at HWE, and gametic equilibrium(Milgroom, 1996; McDonald & Linde, 2002). Theexpectation based on earlier analyses of rice-infectingpopulations from China (Bernardes de Assis et al., 2009),India (Linde et al., 2005), South America (Gonzalez-Veraet al., 2010) and the United States (Rosewich et al., 1999;Bernardes de Assis et al., 2008) was that populations ofR. solani AG-1 IA from northern Iran would exhibit amixed reproductive mode combining recombination andclonal reproduction. The overall clonal fraction was0Æ57, consistent with significant asexual reproduction.But following clone-correction, most of the loci con-formed to HWE expectations (Table 3), consistent withsexual reproduction. High levels of gametic equilibrium(GE) provided additional evidence for recombination inTonekabon (94% of locus pairs in GE) and Amol (93%).In contrast, gametic equilibrium was intermediate inRasht (58% of locus pairs in GE) in the first collection,but increased to 92% in the second collection. Thus allthree Iranian populations of R. solani AG-1 IA exhibit amixed reproductive mode. No inbreeding was detected inany of the populations, suggesting that the pathogen israndom mating in Iran.

(a) (b)

Figure 2 STRUCTURE inferred membership coefficient for multilocus microsatellite genotypes of Rhizoctonia solani AG-1 IA from rice-infecting

population samples obtained in northern Iran at early booting (a) and mature grain (b) rice growth stages. Clusters of individuals based on

prior-defined populations are represented by distinct colours. Each vertical bar represents one individual multilocus genotype. Each colour

represents the most likely ancestry of the cluster from which the genotype or partial genotype was derived. Individuals with multiple colours

have admixed genotypes from k = 3 populations. The bar length indicates its membership coefficient (Q_

) to the distinctly coloured

populations. Statistically significant admixture for an individual multilocus genotype is noted with an asterisk for each individual genotype and

the proportion of admixed genotypes for each population are indicated along the top (***P £ 0Æ001, **P £ 0Æ01 and *P £ 0Æ05). AD is the

percentage of admixture.

56 F. Padasht-Dehkaei et al.

The data here indicate a recombining population struc-ture with some clonality in all three field populations atthe early growth stage. While it is believed that asexualpropagules (i.e. mycelia and sclerotia) are probably themain source of primary infection, it is hypothesized thatthe evidence for recombination at the early stages of theepidemic reflects sexual recombination that occurred inthese populations in previous years. If basidiospores con-tribute significant inoculum to the epidemic during thegrowing season, it is expected that clonality will decreaseand the populations will show increased levels of recom-bination between the early and late stages of the epi-demic. One line of evidence supporting this hypothesiswould be an increase in genotypic diversity between thefirst and the second time points. While clonalitydecreased significantly over time in Tonekabon, in Rashtthere was a significant increase in clonality over time.Using the Psex-value test statistic (Stenberg et al., 2003)the likelihood that the nine strains observed more thanonce in the Rasht population were, in fact, the result ofsexual reproduction was calculated. With Psex valuesranging from 7Æ4 · 10)7 to 7Æ9 · 10)40, it is unlikely thatthese shared genotypes are the result of sexual reproduc-tion. Because average disease incidence and severity inRasht rice fields are lower than in Amol and Tonekabon

fields (Torabi & Binesh, 1984), it is possible that environ-mental conditions between early and late growth stageswere not as favourable for sexual reproduction in Rasht.

Although the immediate effects of sexual reproductionon genotypic diversity may be region dependent, twoother lines of evidence support the importance of basidio-spore secondary inoculum in all three populations. First,a significant decrease in the proportion of admixed geno-types was detected from the early to the late season(P = 0Æ009, Table 3). Because detection of populationadmixture is based on departures from expected HWEproportions in each population, the interpretation is thatthe initially admixed (probably migrant) genotypesrecombined with the resident genotypes during the grow-ing season. Secondly, a significant increase was found inthe proportion of loci under HWE from the early to lateseason (P = 0Æ002, Table 3). It was expected that GDwould decay from the early to the late season as a result ofsexual recombination, but changes in GD were not con-sistent across the three populations. Because IA (a mea-sure of multilocus GD) was highly significant for all threepopulations at both time points, pairwise measures ofGD were not as sensitive as HWE as an indicator of short-term effects due to sexual recombination. An alternativeexplanation for the reduced admixture proportion

Plant Pathology (2013) 62, 49–58

Population genetics of R. solani AG-1 IA from Iran 57

detected later in the season could be that some of theadmixed (probably migrant) genotypes were not as fit asthe endemic ones.

Based on these findings, it is proposed that basidiosp-ores may be a significant source of secondary inoculumduring the development of sheath blight epidemics innorthern Iran and probably in rice fields around theworld. This hypothesis based on a detailed populationgenetic analysis complements previous reports docu-menting the sexual stage from Iran (Khosravi et al., 2011)and Japan (Hashiba & Kobayashi, 1996), which repre-sent different climatic conditions. In both studies, basid-iospore formation in rice fields was observed during theboot stage. It is further proposed that novel pathogengenotypes may be produced by recombination events thatoccur during the middle of the growing season in Iranianrice-infecting populations of R. solani AG-1 IA. Thenovel genotypes that are favoured by selection canincrease in frequency via asexual reproduction withineach field, spreading as clones across short distancesthrough mycelium or sclerotia, and over long distancesvia contaminated seeds, rice straw and equipment.According to the risk model proposed by McDonald &Linde (2002), these characteristics place R. solani AG-1IA among the pathogens with the highest evolutionarypotential, indicating that control based on systemic fungi-cides and major resistance genes should be implementedcarefully to avoid the emergence of fungicide resistanceor virulence.

Acknowledgements

FPD acknowledges the Iranian Ministry of Science,Research and Technology, for supporting him with aresearch assistantship during his PhD at the University ofTehran and a leave of absence to carry out research atETH Zurich from September 2008 to March 2009. Thestatistical analyses on migration patterns were carriedout using the Computational Biology Service Unit fromCornell University (http://cbsuapps.tc.cornell.edu/index.aspx), which is partially funded by Microsoft. Themicrosatellite data were collected using the facilities ofthe Genetic Diversity Center at the ETH. This work waspartially funded by an ETH grant (TH-16 ⁄ 06-1) and aresearch fellowship (Pq-2 308394 ⁄ 2009-7) to PCC fromCNPq (Brazilian National Council for Scientific andTechnological Development).

References

Agapow P-M, Burt A, 2001. Indices of multilocus linkage

disequilibrium. Molecular Ecology Notes 1, 101–2.

ASID, 1998. Cereals: a Statistical View (1988 ⁄ 97). Tehran, Iran:

ASID - Agricultural Statistics and Information Dept., Ministry of

Jihad-e- Agriculture.

Beerli P, Felsenstein J, 2001. Maximum likelihood estimation of a

migration matrix and effective population sizes in n

subpopulations by using a coalescent approach. Proceedings of

the National Academy of Sciences, USA 98, 4563–8.

Plant Pathology (2013) 62, 49–58

Bernardes de Assis J, Peyer P, Rush MC, Zala M,

McDonald BA, Ceresini PC, 2008. Divergence between

sympatric rice- and soybean-infecting populations of

Rhizoctonia solani anastomosis group-1 IA.

Phytopathology 98, 1326–33.

Bernardes de Assis J, Storari M, Zala M et al., 2009. Genetic

structure of populations of the rice-infecting pathogen

Rhizoctonia solani AG-1 IA from China. Phytopathology 99,

1090–9.

Binesh H, Torabi M, 1985. Mode of transmission of rice sheath

blight through seeds and reaction of rice cultivars to the disease.

Iranian Journal of Plant Pathology 21, 15–25.

Ciampi MB, Meyer MC, Costa MJN, Zala M, McDonald BA,

Ceresini PC, 2008. Genetic structure of populations of

Rhizoctonia solani anastomosis group-1 IA from soybean in

Brazil. Phytopathology 98, 932–41.

Cu RM, Mew TW, Cassman KG, Teng PS, 1996. Effect of sheath

blight on yield in tropical, intensive rice production system. Plant

Disease 80, 1103–8.

El Mousadik A, Petit RJ, 1996. High level of genetic differentiation

for allelic richness among populations of the argan tree [Argania

spinosa (L.) Skeels] endemic to Morocco. Theoretical and

Applied Genetics 92, 832–9.

Gonzalez-Vera AD, Bernardes de Assis J, Zala M et al., 2010.

Divergence between sympatric rice- and maize-infecting

populations of Rhizoctonia solani AG 1 IA from Latin America.

Phytopathology 100, 172–82.

Grunwald NJ, Goodwin SB, Milgroom MG, Fry WE, 2003.

Analysis of genotypic diversity data for populations of

microorganisms. Phytopathology 93, 738–46.

Gunnell PS, Webster RK, 1984. Aggregate sheath spot of rice in

California. Plant Disease 68, 529–31.

Hartl DL, Clark AG, 1997. Principles of Population Genetics.

Sunderland, MA, USA: Sinauer Associates.

Hashiba T, Kobayashi T, 1996. Rice diseases incited by

Rhizoctonia species. In: Sneh B, Jabaji-Hare S, Neate S, Dijst G,

eds. Rhizoctonia Species: Taxonomy, Molecular Biology,

Ecology, Pathology and Disease Control. Dordrecht, The

Netherlands: Kluwer Academic Publishers, 331–40.

IRRI, 2002. Standard Evaluation System for Rice: Reference

Guide. Los Banos, The Philippines: IRRI - International Rice

Research Institute.

Johanson A, Turner HC, Mckay GJ, Brown AE, 1998. A PCR-

based method to distinguish fungi of the rice sheath-blight

complex, Rhizoctonia solani, R. oryzae, and R. oryzae-sativae.

FEMS Microbiology Letters 162, 289–94.

Jones RK, Belmar SB, 1989. Characterization and pathogenicity of

Rhizoctonia spp. isolated from rice, soybean, and other crops

grown in rotation with rice in Texas. Plant Disease 73,

1004–10.

Khosravi V, Padasht-Dehkaei F, Naeimi S, Rostami M, Ceresini PC,

2011. First report of naturally occurring Thanatephorus

cucumeris (anamorph: Rhizoctonia solani AG-1 IA) on paddy-

rice fields from Iran. Iranian Journal of Plant Pathology 47,

27–8.

Kobayashi T, Mew TW, Hashiba T, 1997. Relationship between

incidence of rice sheath blight and primary inoculum in the

Philippines: mycelia in plant debris and sclerotia. Annals of the

Phytopathological Society of Japan 63, 324–7.

Kozaka T, 1975. Sheath blight in rice plants and its control.

Review of Plant Protection Research 8, 69–80.

58 F. Padasht-Dehkaei et al.

Lanoiselet VM, Cother EJ, Ash GJ, 2007. Aggregate sheath spot

and sheath spot of rice. Crop Protection 26, 799–808.

Lee FN, Rush MC, 1983. Rice sheath blight: a major rice disease.

Plant Disease 67, 829–32.

Linde CC, Zala M, Paulraj RSD, McDonald BA,

Gnanamanickam SS, 2005. Population structure of the rice

sheath blight pathogen Rhizoctonia solani AG-1 IA from India.

European Journal of Plant Pathology 112, 113–21.

Matsumoto M, 2002. Trials of direct detection and

identification of Rhizoctonia solani AG 1 and AG 2 subgroups

using specifically primed PCR analysis. Mycoscience 43,

185–9.

McDonald BA, Linde C, 2002. Pathogen population genetics,

evolutionary potential, and durable resistance. Annual Review

of Phytopathology 40, 349–79.

Milgroom MG, 1996. Recombination and the multilocus structure

of fungal populations. Annual Review of Phytopathology 34,

457–77.

Milgroom MG, Fry WE, 1997. Contributions of population

genetics to plant disease epidemiology and management.

Advances in Botanical Research 24, 1–30.

Milgroom MG, Peever TL, 2003. Population biology of plant

pathogens: the synthesis of plant disease epidemiology and

population genetics. Plant Disease 87, 608–17.

Mousanezhad S, Alizadeh A, Asafaei N, 2010. Assessment of yield

loss due to rice blast disease in Iran. Journal of Agricultural

Science and Technology 12, 357–64.

Nei M, 1978. Estimation of average heterozygosity and

genetic distance from a number of individuals. Genetics 89,

538–90.

Ogoshi A, 1987. Ecology and pathogenicity of anastomosis and

intraspecific groups of Rhizoctonia solani Kuhn. Annual Review

of Phytopathology 25, 125–43.

Okhovvat SM, 1999. Cereal Diseases (Barley, Wheat, Rice, Corn

and Sorghum). Tehran, Iran: Tehran University Publications.

Ou SH, 1985. Rice Diseases. Farnham Royal, UK: Commonwealth

Agricultural Bureau.

Rosewich UL, Pettway RE, McDonald BA, Kistler HC, 1999. High

levels of gene flow and heterozygote excess characterize

Rhizoctonia solani AG-1 IA (Thanatephorus cucumeris) from

Texas. Fungal Genetics and Biology 28, 148–59.

Savary S, Willocquet L, Teng PS, 1997. Modelling sheath blight

epidemics on rice tillers. Agricultural Systems 55, 359–84.

Savary S, Willocquet L, Elazegui FA et al., 2000. Rice pest

constraints in tropical Asia: characterization of injury profiles

in relation to production situations. Plant Disease 84,

341–56.

Sivalingam PN, Vishwakarma SN, Singh US, 2006. The role of

seed-borne inoculum of Rhizoctonia solani in sheath blight of

rice. Indian Phytopathology 59, 445–52.

Slaton NA, Cartwright RD, Meng J, Gbur Jr. EE, Norman RJ,

2003. Sheath blight severity and rice yield as affected by nitrogen

fertilizer rate, application method, and fungicide. Agronomy

Journal 95, 1489–96.

Stenberg P, Lundmark M, Saura A, 2003. MLGSIM: a program for

detecting clones using a simulation approach. Molecular

Ecology Notes 3, 329–31.

Stevens-Johnk J, Jones RK, 1994. Comparison of whole-cell fatty

acid compositions in intraspecific groups of Rhizoctonia solani

AG-1. Phytopathology, 84, 271–5.

Stoddart JA, Taylor JF, 1988. Genotypic diversity: estimation and

prediction in samples. Genetics 118, 705.

Tajickghanbari MA, Rahimian H, Alizadeh A, 2002. Studies on

diversity of populations of Rhizoctonia solani AG-I-IA in rice

fields of northern Iran. Iranian Journal of Plant Pathology 38,

63–83.

Tajickghanbari MA, Rahimian H, Alizadeh A, 2005. Studies of

populations of Rhizoctonia solani AG-1 IA isolated from rice by

rDNA RFLP in Mazandaran Province. Iranian Journal of Plant

Pathology 41, 204–22.

Torabi M, Binesh H, 1984. Sheath blight disease of rice, study on

causal organism, distribution and susceptibility of some rice

cultivars in north provinces of Iran. Iranian Journal of Plant

Pathology 20, 6–8.

Weir BS, Cockerham CC, 1984. Estimating F-statistics for the

analysis of population structure. Evolution 38, 1358–70.

Willocquet L, Fernandez L, Savary S, 2000. Effect of various crop

establishment methods practised by Asian farmers on epidemics

of rice sheath blight caused by Rhizoctonia solani. Plant

Pathology 49, 346–54.

Yang XB, Snow JP, Berggren GT, 1989. Morphogenesis of

microsclerotia and sasakii-type sclerotia in Rhizoctonia solani,

anastomosis group 1, intraspecific groups IA and IB.

Mycological Research 93, 429–34.

Zala M, McDonald BA, Bernardes de Assis J et al., 2008. Highly

polymorphic microsatellite loci in the maize- and rice-infecting

fungal pathogen Rhizoctonia solani anastomosis group 1 IA.

Molecular Ecology Resources 8, 686–9.

Zhan J, Mundt CC, McDonald BA, 1998. Measuring immigration

and sexual reproduction in field populations of Mycosphaerella

graminicola. Phytopathology 88, 1330–7.

Zhang Q, 2007. Strategies for developing green super rice.

Proceedings of the National Academy of Sciences, USA 104,

16402–9.

Plant Pathology (2013) 62, 49–58