First Report of Fusarium oxysporum f.sp. pisi as Causal Agent of Root and Crown Rot on Chickpea (Cicer arietinum) in Southern Italy.

Epidemiological investigations in representative chickpea (Cicer arietinum L.) fields in southern Italy (Larino, Campobasso, 41°50'45″ N, 14°55'28″ E) identified severe withering (25 to 51%) of plants during flowering. Diseased plants showed a reduced total root biomass associated with less vigorous and chlorotic foliage. Browning and necrosis of subcortical and xylematic tissues of the crown and main roots were observed in affected plants. Symptomatic root and stem portions from 50 plants were sampled, surface disinfected with a sodium hypochlorite water solution (2% v/v for 2 min), rinsed with sterile distilled water, and placed in petri dishes containing potato dextrose agar with streptomycin sulfate (200 mg/l) and incubated at 25°C for 10 days. The most frequent fungal colony isolated showed macro- and microscopic characters specific of the genus Fusarium (3), with falcate and three-septate macroconidia (24.0 to 43.8 µm long) and microconidia (6.8 to 10.4 µm long) with zero or one septa. The ribosomal DNA of the fungal isolate processed by PCR using the ITS1F/ITS4 primers (2) produced an amplicon of 545 bp (ENA, Accession No. HG423346). A BLAST search with the amplified sequence in the database of the International Mycological Association ( www.mycobank.org ) revealed 99% identity with F. oxysporum sequences. Additional molecular analysis using the specific primers Foc0-12/Foc0-12rf for F. oxysporum f.sp. ciceris (Foc) produced an amplicon only in the chickpea virulent strain Foc-7952, race 0 (1) used as control; furthermore, PCR amplification for the Pisatin Demetylase gene by using the specific primers PDAF2a and PDAR3a (4) yielded the expected amplicon only for the new isolate, whereas no amplification was obtained with the control strain Foc-7952. Pathogenicity assays were carried out to complete Koch's postulates. To this aim, horticultural peat was infested with a conidial suspension (1 × 104 conidia/g of soil) from the new fungal pathogen, dispensed in plastic pots, and sown with surface sterilized seeds of chickpea (cv. Real, ISEA, Italy). Uncontaminated peat was used as control. For both treatments, 3 replicates of 10 seeds were used and experiments repeated twice. The plastic pots were kept in a growth chamber (28°C; 70% RH; 15/9 h light/dark) where the first disease symptoms on plants appeared 20 days after sowing. At the end of the experiments, all plants inoculated with the new isolate showed a high disease severity (98%), whereas non-inoculated plants remained healthy. The seedlings from infested soil demonstrated the same symptoms previously observed in the field, and after re-isolation, the causal agent demonstrated the same morphological features of the isolate used for inoculation. Pathogenicity tests were performed on pea, faba bean, melon, and tomato by using three cultivars for each crop. The results demonstrated high virulence of the new isolate of F. oxysporum f.sp. pisi (Fop) on both chickpea and pea with seed germination reduction, rot on main and secondary roots and cotyledonary leaves, and root biomass reduction and foliage chlorosis. No symptoms were observed on other inoculated vegetal species. Collectively, data of our investigation allow us to affirm that this is the first report of Fop as a new pathogen of chickpea. This result has great economic importance since it enables specific monitoring and management plans for this new disease caused by Fop on chickpea, a key crop for human and animal nutrition. References: (1) M. M. Jiménez-Gasco and R. M. Jiménez-Díaz, Phytopathology 93:201, 2003. (2) I. Larena et al. J. Biotechnol. 75:187, 1999. (3) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (4) N. A. Milani et al. Fungal Genet. Biol. 933:942, 2012.

Environmental factors affect the activity of biocontrol agents against ochratoxigenic Aspergillus carbonarius on wine grape.

The influence of temperature and relative humidity (RH) on the activity of three biocontrol agents-the yeast Metschnikowia pulcherrima LS16 and two strains of the yeast-like fungus Aureobasidium pullulans LS30 and AU34-2-against infection by A. carbonarius and ochratoxin A (OTA) accumulation in wine grape berries was investigated in lab-scale experiments. The presence of wounds on grape skin dramatically favored infection of berries by A. carbonarius strain A1102, since unwounded berries showed very low levels of infection at all conditions of RH and temperature tested. Artificially wounded berries pre-treated with the biocontrol agents were inoculated with the ochratoxigenic A. carbonarius strain A1102 and were incubated for 5 days at two levels of RH (60% and 100%) and three different temperatures (20, 25 and 30 °C). The three biocontrol agents were able to prevent infections at 60% RH and 20 °C. At 60% RH and 25 °C only strain AU34-2 achieved some protection on day 5, whereas at 30 °C a limited biocontrol efficacy was evident only up to day 2. At 100% RH, LS16, LS30 and AU34-2 showed effective protection of grape berries at 20 °C until the 5th day of incubation. The three biocontrol agents achieved significant protection at higher temperatures only until the 2nd day after the beginning of the experiment: all three strains at 25 °C, and only strain LS16 at 30 °C. After 5 days, the three biocontrol agents were able to significantly reduce the level of OTA in berries at all the conditions tested. This occurred even when protection from infection was not significant, except at 30 °C and 100% of RH for all the three strains, and at 25 °C and 100% of RH for strain LS16. The biocontrol agents displayed a higher rate of colonization on grape berries at 20 and 25 °C than at 30 °C. The higher value of RH (100%) appeared to increase the rate of colonization, in particular at 20 and 25 °C. Taken together, our results emphasize the significant influence of environmental factors on the effectiveness of biocontrol against A. carbonarius as well as on OTA contamination in wine grape berries, and the need for biocontrol agents that can cope with the environmental conditions that are conducive to attack by A. carbonarius.

Strains of Aureobasidium pullulans can lower ochratoxin A contamination in wine grapes.

Wine contamination with ochratoxin A (OTA) is due to the attack of wine grapes by ochratoxigenic Aspergillus carbonarius and Aspergillus spp. section Nigri. Four A. pullulans strains, AU14-3-1, AU18-3B, AU34-2, and LS30, are resistant to and actively degrade ochratoxin A in vitro. The less toxic ochratoxin alpha and the aminoacid L-beta-phenylalanine were the major degradation products, deriving from the cleavage of the amide bond linking these two moieties of OTA. The same strains were studied further as biocontrol agents of A. carbonarius on wine grapes in laboratory experiments. Three of the four strains significantly prevented infections by A. carbonarius. Berries pretreated with the biocontrol agents and infected with A. carbonarius contained lower amounts of OTA as compared to the untreated infected control berries. Two of these strains were shown to degrade OTA to ochratoxin alpha in fresh grape must, but the mechanisms of the decrease of OTA accumulation in infected berries pretreated with the biocontrol agents remain to be elucidated. Assessment of one strain carried out in the vineyard during the growing season of 2006 showed that the tested strain was an effective biocontrol agent, reducing both severity of Aspergillus rots and OTA accumulation in wine grapes. To our knowledge this is the first report describing the positive influence of biocontrol agents on OTA accumulation in this crop species.

Integration of biocontrol agents and food-grade additives for enhancing protection of stored apples from Penicillium expansum.

Forty-nine compounds currently used as additives in foods were tested in combination with three biocontrol agents, the yeasts Rhodotorula glutinis, Cryptococcus laurentii, and the yeastlike fungus Aureobasidium pullulans, to increase their antagonistic activity against Penicillium expansum, the causal agent of blue mold on apples. Twelve additives dramatically improved the antagonistic activity of one or more of the tested biocontrol agents. In a two-way factorial experiment with these selected additives the percentage of P. expansum rots on apples was significantly influenced by the antagonist and the additive as well as by their interaction. The combination of the biocontrol agents and some additives resulted in a significantly higher activity with respect to the single treatments applied separately, producing additive or synergistic effects. Some of the selected additives combined with a low yeast concentration (106 cells per ml) had comparable or higher efficacy than the biocontrol agents applied alone at a 100-fold higher concentration (10(8) cells per ml). Some organic and inorganic calcium salts, natural gums, and some antioxidants displayed the best results. In general, the effect of each additive was specific to the biocontrol isolate used in the experiments. Possible mechanisms involved in the activity of these beneficial additives and their potential application in effective formulations of postharvest biofungicides are discussed.