Genetic characteristics of Fusarium verticillioides from corn in the Philippines

J Gen Plant Pathol (2009) 75:405 412 DOI 10.1007/s10327-009-0199-4 FUNGAL DISEASES Genetic characteristics of Fusarium verticillioides from corn in the Philippines Christian Joseph R. Cumagun Josephine S. Ramos Arvin O. Dimaano Françoise Munaut François Van Hove Received: 7 May 2009 / Accepted: 11 August 2009 / Published online: 24 October 2009 Ó The Phytopathological Society of Japan and Springer 2009 Abstract Fusarium verticillioides (teleomorph: Gibberella moniliformis = G. fujikuroi mating population A) is one of the most important fungal pathogens of corn worldwide. The pathogen produces fumonisins, mycotoxins that are potentially harmful to humans and animals. Thirty-five Fusarium isolates from Laguna and Isabela, Philippines were identified morphologically and molecularly as F. verticillioides and characterized by PCR for mating type (MAT). Twenty-six isolates were MAT1-2, while nine isolates were MAT1-1. The isolates from Isabela were tested for aggressiveness, rated according to a disease index (%) on Super Sweet corn IPB variety 1 under field conditions across two trials using the toothpick inoculation method. Other aggressiveness traits such as inhibition of seedling emergence, decrease of seedling height, fresh and dry mass were also determined in two greenhouse trials. All isolates were pathogenic to corn seedlings and mature plants compared to the noninoculated control. Significant genotypic variation was observed (P = 0.01) in trial, isolate, and isolate 9 trial interaction for all traits across two greenhouse trials and that aggressiveness was highly influenced by the trial conditions. Similarly, significant genotypic variation was observed in trial, replication within trial and isolate 9 trial interaction. Heritability was high C. J. R. Cumagun (&) J. S. Ramos A. O. Dimaano Crop Protection Cluster, College of Agriculture, University of the Philippines Los Baños (UPLB), College, 4031 Laguna, Philippines e-mail: christian_cumagun@yahoo.com F. Munaut F. Van Hove Université Catholique de Louvain, Unité de Microbiologie (MBLA), Mycothèque de l Université Catholique de Louvain (BCCM TM /MUCL), Croix du Sud, 3 box 6, 1348 Louvain-la Neuve, Belgium for the five traits in the greenhouse (h 2 = 0.80 0.90) but moderate for disease index in the field (h 2 = 0.49). In an analysis of fumonisin production in corn culture by high performance liquid chromatography, 30 of 35 strains produced a detectable level of fumonisins, varying from 0.44 to 742 lg FB 1 /g corn, 0.51 222 lg FB 2 /g and 0.12 37 lg FB 3 /g. Isabela isolates produced more fumonisins than the Laguna isolates did. In vitro fumonisin production had little correlation with the field disease index (r = 0.32) or with greenhouse seedling germination (0.25). Keywords Aggressiveness Corn F. verticillioides Fumonisins Mating type Introduction Fusarium verticillioides (Saccardo) Nirenberg (teleomorph: Gibberella moniliformis Wineland, = G. fujikuroi mating population A) is a major pathogen of corn worldwide (Nelson 1992; Sharma et al. 1993) and is the most frequently reported Fusarium species in corn (Leslie 1991). The fungus was first reported in the Philippines by Reinking in 1918 in corn ears and is also causing seedling, stalk, and ear rots (Marasas et al. 1984; Nelson 1992). F. verticillioides is common in farm and storage samples in various parts of the Philippines (Esteves et al. 1996). The pathogen can cause stand reduction and poor kernel quality and is a potential threat to human and animal health as a result of mycotoxins such as moniliformin, fusarin C, fusaric acid and fumonisins that it produces (Desjardins 2006). Eleven different mating populations have been recognized in the G. fujikuroi species complex (Lepoint et al. 2005; Leslie 1991, 1999; Phan et al. 2004), and three of these 11 mating populations, designated A, D, and E, are

406 J Gen Plant Pathol (2009) 75:405 412 pathogens of corn (Munkvold and Desjardins 1997). G. fujikuroi mating population A is heterothallic (self-sterile), thus enabling genetic crosses of two different mating types (Fincham et al. 1979; Leslie and Summerell 2006). In the Philippines, little work has been done to characterize isolates of F. verticillioides with respect to mating type, aggressiveness, and fumonisins although the pathogen was first reported almost a century ago (Reinking 1918). Fumonisins were reported in corn in the southern Philippines for the first time in the 1990s (Miller et al. 1993), but only a few succeeding studies such as those by Rallos and Dalmacio (2004) and Yamashita et al. (1995) are available. We presume that most of the isolates on corn in the Philippines belong to the fumonisin-producing mating population. Knowledge of genetic variation in important fitness traits such as fertility, aggressiveness, and fumonisin production of Fusarium spp. in corn is important in resistance breeding, the evolutionary risk of the pathogen and the design of agricultural management practices (Cumagun 2004; Leslie 1991). The objectives of this study are (1) to characterize the mating types and fumonisin production in a population of F. verticillioides from Laguna and Isabela provinces and (2) to determine the genetic variation in aggressiveness among strains found in Isabela under greenhouse and field conditions. Materials and methods Isolation and maintenance of cultures Healthy and moldy corn cobs were collected from the Institute of Plant Breeding, UPLB and corn fields in Calamba Laguna (southern Luzon) and in several barangays in Isabela (northern Luzon) Philippines. These two provinces were chosen because Isabela is the top cornproducing province in the Philippines and Laguna is a small-scale corn-growing province. Kernels were surfaced sterilized with 1% (w/v) NaOCl, rinsed in three changes of sterile distilled water, blotted dry with tissue paper and plated on pentachloronitrobenzene (PCNB) selective medium (Nash and Snyder 1962). After the cultures were incubated in the light for 4 5 days, the typical pinkish growth on the media was subcultured. Morphological identification was based on the identification manual by Leslie and Summerell (2006). Molecular identification was based on the sequencing of the elongation factor (EF-1a) gene as described in O Donnell et al. (1998). All singlespored strains labeled with a Mycothèque de l Université catholique de Louvain (MUCL) prefix are stored for longterm conservation on potato dextrose agar (PDA) and synthetic nutrient agar (SNA) slants, in lyophilized form and by cryopreservation ( 130 C), at the Mycothèque de l Université catholique de Louvain (BCCM TM /MUCL) culture collection (Table 1). Crossing and mating type PCR Isolates were single-spored before use in sexual crosses. The mating type of 35 isolates was determined by crossing as described by Klittich and Leslie (1988) except for the use of Tween 80. Standard tester strains FGSC 7416 (MATA-2) and FGSC 7415 (MATA-1) were first used as female parents (Fungal Genetics Stock Center, Missouri, USA). Reciprocal crosses were done to determine female fertility of the 35 isolates. A fertile cross is one in which a cirrhus of ascospores can be seen emerging from a mature perithecium 2 4 weeks after fertilization. All crosses were repeated twice. The mating type of 35 isolates was determined by a mating-type PCR (MAT-PCR) as described by Steenkamp et al. (2000). Greenhouse aggressiveness test Seeds of the Super Sweet corn IPB variety 1 were disinfected in sterile distilled water at room temperature for 4h,in55 C water for 5 min, then in cool water for a few seconds, followed by air-drying overnight (Desjardins et al. 1998). Seventeen isolates of F. verticilioides from Isabela were cultivated on PDA with 12 h continuous light for 5 7 days. A conidial suspension was then prepared by flooding the petri plate with 10 ml of sterile distilled water. Fungal growth was scraped-off using a spatula. The suspension was filtered through several layers of cheesecloth, and the concentration was adjusted to 10 5 conidia per ml using a haemacytometer. Seeds were soaked in 0.5 % (w/v) NaOCl solution for 1 min and rinsed twice with sterile distilled water. Seeds were then soaked in spore suspension at ambient temperature overnight and then allowed to dry before sowing in 15 cm diameter pots. Five replicates of 15 seeds were sown in autoclaved soil. The conditions of the greenhouse in two trials ranged from 27 to 28 C and 82 to 85% relative humidity. The isolate aggressiveness was scored on the basis of the percentage of seed germination and, for all seedlings at 14 days after sowing, the decrease in height (distance from soil to highest leaf), fresh mass (whole plant including roots), and dry mass after 48 h at 40 C (when there was no further change in plant biomass). Noninoculated plants were used as controls, and tests were repeated twice (July and August 2005). Field aggressiveness test Corn stalks were inoculated with one of the 17 Fusarium isolates (Table 2) using the toothpick technique (Jardine

J Gen Plant Pathol (2009) 75:405 412 407 Table 1 Origin and genetic characteristics of 35 strains of Fusarium verticillioides from corn in the Philippines MUCL no. a Origin EF-1a sequences b Mating type MAT-PCR Fumonisin production (lg/g) c B 1 B 2 B 3 49886 Los Baños, Laguna F. verticillioides (99.2%) MATA-2 MAT1-2 d 82 22 4.5 49887 Calamba, Laguna F. verticillioides (99.2%) MATA-2 MAT1-2 d 19 2.3 ND 49889 Calamba, Laguna F. verticillioides (99.2%) MATA-2 MAT1-2 ND e ND ND 49890 Los Baños, Laguna F. verticillioides (99.2%) MATA-2 MAT1-2 ND ND ND 49891 Los Baños, Laguna F. verticillioides (99.2%) MATA-2 MAT1-2 d ND ND ND 49892 Los Baños, Laguna F. verticillioides (99.3%) MATA-1 MAT1-1 d 18 ND ND 49894 Los Baños, Laguna F. verticillioides (99.2%) MATA-2 MAT1-2 101 27 3.2 49895 Los Baños, Laguna F. verticillioides (99.2%) MATA-2 MAT1-2 0.44 ND ND 49897 Los Baños, Laguna F. verticillioides (99.2%) MATA-2 MAT1-2 d 5.0 0.52 0.16 49898 Los Baños, Laguna F. verticillioides (99.2%) MATA-1 MAT1-1 ND ND ND 49899 Los Baños, Laguna F. verticillioides (99.2%) MATA-2 MAT1-2 d 1.2 ND ND 49901 Los Baños, Laguna F. verticillioides (99.2%) MATA-2 MAT1-2 d 16 3.9 0.39 49902 Los Baños, Laguna F. verticillioides (99.2%) MATA-2 MAT1-2 1.9 0.51 ND 49904 Los Baños, Laguna F. verticillioides (99.2%) MATA-2 MAT1-2 8.5 1.2 0.32 51055 Casibabarag, Isabela F. verticillioides (99.7%) MATA-2 MAT1-2 d 382 87 17 51056 Catabayungan, Isabela F. verticillioides (99.6%) f MAT1-2 25 39 ND 51057 Casibabarag, Isabela F. verticillioides (99.7%) MATA-1 MAT1-1 99 5.7 23 51058 Catabayungan, Isabela F. verticillioides (99.3%) f MAT1-2 79 15 4.6 51059 Catabayungan, Isabela F. verticillioides (99.3%) MATA-2 MAT1-2 d 4.1 ND ND 51060 Casibabarag, Isabela F. verticillioides (99.2%) f MAT1-1 19 2.6 0.93 51061 Casibabarag, Isabela F. verticillioides (99.2%) MATA-1 MAT1-1 19 4.5 0.45 51062 Catabayungan, Isabela F. verticillioides (99.7%) MATA-2 MAT1-2 d 647 108 37 51063 Catabayungan, Isabela F. verticillioides (99.7%) MATA-1 MAT1-1 ND ND ND 51064 Casibabarag, Isabela F. verticillioides (99.7%) MATA-2 MAT1-2 d 137 15 8.8 51066 San Pablo, Isabela F. verticillioides (99.6%) f MAT1-2 d 86 18 2.9 51067 San Pablo, Isabela F. verticillioides (99.6%) MATA-2 MAT1-2 2.0 ND ND 51068 San Pablo, Isabela F. verticillioides (99.6%) MATA-2 MAT1-2 d 38 6.0 1.3 51069 San Pablo, Isabela F. verticillioides (99.7%) MATA-1 MAT1-1 742 204 24 51070 San Pablo, Isabela F. verticillioides (99.6%) f MAT1-2 290 222 25 51071 San Pablo, Isabela F. verticillioides (99.7%) MATA-1 MAT1-1 d 2.0 ND 0.12 51072 Santa Maria, Isabela F. verticillioides (99.7%) MATA-2 MAT1-2 d 26 2.2 2.0 51073 Santa, Maria, Isabela F. verticillioides (99.7%) MATA-2 MAT1-2 43 6.0 2.9 51074 Santa, Maria, Isabela F. verticillioides (99.6%) MATA-2 MAT1-2 159 15 7.5 51075 Casibabarag, Isabela F. verticillioides (99.6%) f MAT1-1 107 19 5.3 51076 Los Baños, Laguna F. verticillioides (99.2%) MATA-2 MAT1-2 128 17 6.4 a MUCL no., code of the strain cultures preserved in the Mycothèque de l Université catholique de Louvain (BCCM TM /MUCL) b Percentage similarity of EF-1a gene sequence against that of the F. verticillioides reference strain FGSC_7600 (FRC M3125, KSU A00149, MUCL 43478) available at the Broad Institute (http://www.broad.mit.edu) c Analysed by HPLC: limit of detection (LOD) (signal/noise = 3) for fumonisins B 1,B 2 and B 3 was 0.0076, 0.012 and 0.0026 lg of fumonisin/g of corn, respectively; limit of quantification (LOQ) (signal/noise = 10) was 0.025 lg/g for B 1, 0.041 lg/g for B 2 and 0.0085 lg/g for B 3 d Female fertile e ND not detected (below LOD) f Negative cross with the standard tester strain and Leslie 1992). Round wooden toothpicks were boiled twice in distilled water for 1 h to remove any toxic substances that might inhibit growth of the Fusarium isolates (Jardine and Leslie 1999; Asran and Buchenauer 2002). The toothpicks were then placed in screw-capped test tubes containing 5 ml potato dextrose broth and autoclaved.

408 J Gen Plant Pathol (2009) 75:405 412 Table 2 Aggressiveness in greenhouse and field trials of 17 Fusarium verticillioides isolates from Isabela province Seed inoculation a Toothpick inoculation b MUCL no. Germination (%) Seedling height (cm) Fresh weight (g) Dry weight (g) Disease index (%) 51055 85 11.6* 21.93* 9.83* 50.94 51056 75* 11.3* 18.55* 8.55* 54.76 51057 70* 11.1* 19.44* 8.95* 43.48 51058 64* 9.4* 14.49* 2.40* 34.38 51059 89 11.9* 22.47* 11.31* 33.66 51060 79* 10.9* 18.80* 6.59* 49.43 51061 67* 9.2* 13.39* 3.43* 32.16 51062 82 10.6* 18.87* 8.85* 44.25 51064 82 10.6* 19.19* 8.84* 30.70 51066 58* 10.4* 15.43* 3.34* 34.49 51067 68* 11.0* 18.27* 6.22* 41.25 51068 82 11.8* 21.70* 10.69* 34.64 51069 71* 10.6* 17.92* 5.64* 45.12 51071 55* 8.9* 12.75* 2.18* 40.38 51072 59* 9.2* 13.71* 2.06* 37.21 51073 73* 10.5* 20.09* 9.72* 36.91 51074 64* 9.6* 14.70* 2.98* 38.67 Population mean 72 11.0 17.75 6.56 40.14 Control 93 13.8 25.89 15.79 c LSD5% 14.14 1.19 2.80 3.44 15.04 * Statistically different from the control at 5% level significance; LSD least significant difference a Germination percentage is based on 150 seedlings (15 seedlings/replicate, 5 replicates/trial, 2 trials) grown for 15 days in sterile soil in the greenhouse. Seedling height, fresh mass and dry mass are the averages of all the germinated seedlings from 150 initial seeds per isolate. Data on germination, seedling height, fresh mass and dry mass were compared with the control at 5% level b Measurement is based on length of lesion produced in corn stalks (average of 45 plants/season) in the field after inoculation with F. verticillioides-infested toothpicks c Not determined Upon cooling, agar disks of F. verticillioides were placed into the test tubes and incubated at room temperature for 10 days under continuous light. Plants grown in the field were inoculated at the flowering stage, 60 days after planting, by creating a hole in the second internode of the stalk using an ice pick disinfected with 10% (w/v) NaOCl. One of the toothpicks was then inserted into the hole. Two months later at harvest, we cut the stalks longitudinally and rated the disease severity. Discoloration and rotting on the inoculated stalks were rated from 1 to 5, where 1 = 0 25%, 2 C 25 50%, 3 C 50 75%, 4 C 75 \100% of the inoculated internodes, and 5 = 100% with infection extending into the adjacent internodes. The formula for obtaining the disease index is as follows: Disease index ð% Þ ¼ ½ð0A þ 1B þ 2C þ 3D þ 4E þ 5FÞ 100Š5T; where A, B, C, D, E, and F are the total number of maize stalks with an index of 0, 1, 2, 3, 4, and 5, respectively, and 5T is the total number of stalks multiplied by the maximum disease rating (Hooker 1956).The experiment was conducted for two seasons: September to October 2007 (wet season) with mean rainfall (7.8 mm), relative humidity (85%) and temperature (27.6 C) and March to April 2008 (dry season) with mean rainfall (1.7 mm), relative humidity (80%) and temperature (28.3 C, otherwise it is the same as wet season) at the Central Experiment Station, UPLB. Mycotoxin analyses The ability of 35 isolates to produce fumonisins (FB) on maize kernels was tested as described by Thiel et al. (1991) and analyzed by high performance liquid chromatography (HPLC) as described by Shephard et al. (1990) and Sydenham et al. (1996). The limit of detection (LOD) (signal/ noise = 3) for FB 1,FB 2 and FB 3 was, respectively, 0.0076, 0.012 and 0.0026 lg of fumonisin/g of corn, and the limit of quantification (LOQ) (signal/noise = 10) was 0.025 lg/g for FB 1, 0.041 lg/g for FB 2 and 0.0085 lg/g for FB 3.

J Gen Plant Pathol (2009) 75:405 412 409 Statistical analyses Experimental design was a randomized complete block design with five replicates in the greenhouse and three replicates per treatment (=isolates) in the field. Means of two trials in the greenhouse and field were compared by a t-test of differences in least-square means. Plot means were used for analysis of variance. Coefficient of variation (CV) (%) of the respective variance components (r 2 ) was calculated according to the formula: CV (%) = (Hr 2 /l) 9 100, where r 2 = variance component and l = population mean. Repeatability estimates were calculated by partitioning the phenotypic variance of spatial replicates with one experiment according to the formula r 2 g/r 2 g? r 2 e (Falconer 1989) wherer 2 g = genotypic variance and r 2 e = error variance. Broad sense heritabilities (h 2 ) were estimated on an entry mean basis (Fehr 1987). Confidence intervals of heritability were computed according to Knapp and Bridges (1987). Estimates of variance components were calculated as described by Snedecor and Cochran (1989). All variables were analyzed with the statistical package PLABSTAT (Utz 2000). Results Thirty-five isolates were identified morphologically as F. verticillioides based on the manual by Leslie and Summerell (2006). In addition, these identifications were confirmed molecularly by sequencing the EF-1a gene (Table 1). Twenty-six isolates were identified as MAT1-2 and nine as MAT1-1 using MAT-PCR, consistent with the mating-type data generated on positive crosses with tester strains (Table 1). The 17 strains of F. verticillioides isolated from naturally infected corn kernels were examined for their aggressiveness on seedlings of Super Sweet corn IPB variety 1 in the greenhouse (Table 2). Germination, seedling height, fresh mass and dry mass differed significantly from those of the control except for germination after treatment with five isolates. In the greenhouse, the most aggressive isolate, 51071, reduced seedling germination to 41% relative to the control, whereas the least aggressive isolate, 51059, reduced seedling stand by 4% relative to the control. In vitro fumonisin production had a low correlation with the field disease index (r = 0.32) and with greenhouse seedling germination (r = 0.25) (Fig. 1). Correlations between the disease index and plant height, fresh mass, and dry mass were also low (data not shown). The isolates varied in their field disease severity in corn stalks, which was interpreted as differences in aggressiveness (Table 2). Mean field disease indices ranged from 30.7 to 54.8% with mean values of 40.14%, respectively. On 35 isolates tested for fumonisin production, 30 produced detectable levels. Isabela isolates produced higher levels than did the Laguna isolates. Fumonisinproductionrangedfrom0.44to742lgFB 1 /g corn, 0.51 to 222 lg FB 2 /g and 0.12 to 37 lg FB 3 /g (Table 1). The amount of fumonisin B 1 produced by 17 F. verticillioides isolates from Isabela province (Table 1) was highly variable (2 647 lg/g). Highly significant trial, isolate, and isolate 9 trial variation was found for the four aggressiveness traits in the greenhouse (Table 3) and for trial, replication within trial, and isolate 9 trial interaction for the field disease index in the field (Table 4). Heritability was also higher in the greenhouse (h 2 = 0.80 0.90) than in the field (h 2 = 0.49). Isolates were the most important source of variation in the in vitro production of the three types of fumonisins, having highly significant differences with high repeatability (Table 5). Discussion Among the 35 isolates of F. verticillioides mating population A analysed in this study, MAT1-2 was more frequent Fig. 1 Correlation between in vitro production of fumonisin B 1 (FB 1 ) and either (a) the field disease index (%) or (b) greenhouse seedling germination for 17 Fusarium verticillioides isolates from Isabela province used to inoculate corn seedlings. Data were combined across two greenhouse and field trials a In vitro FB1 production (ug/g) 800 700 600 500 400 300 200 100 r = 0.32** LSD 5% b In vitro FB1 production (ug/g) 800 700 600 500 400 300 200 100 r = 0.25** LSD 5% 0 0 20 40 60 Field disease index (%) 0 0 50 100 Greenhouse seedling germination (%)

410 J Gen Plant Pathol (2009) 75:405 412 Table 3 Coefficient of variation (%) for germination, plant height, fresh mass and dry mass for 17 isolates of Fusarium verticillioides (I) on the Super Sweet corn IPB variety 1 across two greenhouse trials (T) Parameter df Germination Plant height Fresh mass Dry mass Source of variation Trial (T) 1 8.43** 1.54** 5.50** 18.19** Replication (R):T 8 Isolate (I) 16 12.60** 8.41** 16.66** 46.43** I 9 T 16 6.59** 3.30** 6.48** 24.48** Pooled error 128 13.47 7.16 9.20 18.46 Heritability 0.80 0.87 0.90 0.87 90% CI on h 2a 0.45 0.93 0.64 0.95 0.73 0.97 0.63 0.95 ** Significant at P = 0.01, respectively; -: negative estimate a Confidence intervals (CI) on h 2 were calculated using the method of Knapp and Bridges (1987) Table 4 Coefficient of variation (%) for field disease index for 17 isolates of Fusarium verticillioides (I) on Super Sweet corn IPB variety 1 across two field trials (T) Parameter df Disease index Source of variation Trial (T) 1 36.97** Replication (R):T 4 6.81** Isolate (I) 16 12.13? I 9 T 16 15.57** Pooled error 52 14.48 Heritability 0.49 90% CI on h 2a -0.42 0.81 **,? Significant at P = 0.01 and P = 0.1, respectively. See Materials and methods for description of disease index a Confidence intervals (CI) on h 2 were calculated using the method of Knapp and Bridges (1987) Table 5 Coefficient of variation (%) for in vitro fumonisin B 1,B 2 and B 3 production for 17 isolates of Fusarium verticillioides (I) on Super Sweet corn IPB variety 1 Parameter df Fumonisins B 1 B 2 B 3 Source of variation Replication (R) 2 7.85* 8.16* 8.03* Isolate (I) 16 152.85** 173.69** 127.96** Pooled error 30 18.90 21.16 18.31 Repeatability 98.5 98.5 98.0 **, * Significant at P = 0.01 and P = 0.05, respectively than MAT1-1 (26:9). Whenever sexual crosses fail, MAT- PCR has become very reliable for determining mating type (Leslie and Summerell 2006; Steenkamp et al. 2000). Although the deviation of these results from the expected 1:1 ratio is of low significance, it is worthwhile to note that a larger population of 80 isolates of Fusarium from corn in the Philippines segregated at a Mendelian ratio of 1:1 (Cumagun 2008). Sexual reproduction is a major mechanism generating genotypic variation. For sexual reproduction to occur under field conditions, multiple strains must be present (Kedera et al. 1994). The presence of hermaphrodites could initiate sexual reproduction, which is primarily responsible for the high genotypic diversity. All 17 F. verticilioides isolates from Isabela were pathogenic but varied in their aggressiveness. Significant genotypic variation was observed (P = 0.01) in trial, isolate and isolate 9 trial interaction for all traits across two greenhouse trials. The observed variation in aggressiveness of the 17 F. verticillioides isolates in two greenhouse trials concurs with the findings of Asran and Buchenauer (2002) after corn seedlings were inoculated with 32 F. verticillioides isolates. In the greenhouse, high correlation was found between seedling height, fresh mass, dry mass results and germination percentages (data not shown). This result concurs with the high correlation shown between shoot length and dry mass (r = 0.98) by Danielsen et al. (1998). Germination is the best measure to determine aggressiveness because low germination is a direct effect of the activity of the fungus. Considering that the variation in aggressiveness and fumonisin production was high, an environmental component to this variation could be more important than any genetic component. Indeed, isolates during the two trials/season with a dry season had higher mean aggressiveness (50.53%) than during a wet season (29.76%) (data not shown). There is a dearth of information on mycotoxin production in tropical countries where mycotoxins are a more serious concern. Detection of these mycotoxins could lead to minimizing their levels and promoting food and feed safety. In Southeast Asia, this aspect deserves more attention and priority in research. The reports by Rallos and Dalmacio (2004), Yamashita et al. (1995) and Miller et al.

J Gen Plant Pathol (2009) 75:405 412 411 (1993) are to date the only ones on the occurrence of fumonisins in corn in the Philippines. The present experiment provides evidence that F. verticillioides isolates in corn from Isabela in northern Luzon produced more fumonisins than those isolates from southern Luzon in Laguna. This finding indicates that fumonisins could pose a higher risk of contamination in the primary corn-growing regions such as Isabela than in Laguna where corn is not grown intensively. In the Philippines, phytotoxicity and cytotoxicity of fumonisins were first studied by Rallos and Dalmacio (2004). They reported that a maize isolate of F. verticillioides produced fumonisin B1 up to 13.1 ppm (w/v) in cornmeal medium. The isolate caused stunting of seedlings similar to the stunting observed in our study. In the present study, we revealed that strains of Fusarium mating population A produced fumonisins in Laguna and Isabela corn, representing a risk of fumonisin contamination and thus to the health of humans and farm animals in the Philippines (Chulze et al. 2000; Leslie et al. 1992; Marasas et al. 1984). That all but five of the large number of isolates (N = 35) used in this study produced fumonisins supports this view and earlier findings (Miller et al. 1993; Yamashita et al. 1995). Jardine and Leslie (1999) found no association between stalk rot and the ability to produce fumonisins in vitro, perhaps because fumonisin production in vitro differs from that produced during plant pathogenesis. In another study, in a progeny population from a cross between a fum1? field strain that was highly virulent and a fum1 - field strain that was weakly virulent, high levels of virulence on maize seedlings were associated with those progeny that produced fumonisin, but symptoms were also induced by nonproducers (Desjardins et al. 1995). This finding suggests that fumonisins play a role in virulence, but they are not essential for virulence on maize seedlings. Recently, Glenn et al. (2008) transformed a nonfumonisin-producing strain of F. verticillioides isolated from banana and nonpathogenic to maize to obtain fumonisin-producing transformants that were pathogenic on maize seedlings. This finding indicated that fumonisin production by F. verticillioides is essential for symptom development on maize seedlings. Little effort has been made to breed varieties resistant to Fusarium diseases in the Philippines, and there is no program to breed for resistance. 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