Identification of Species of Botryosphaeriaceae Causing Bot Gummosis in Citrus in California.

Members of the Botryosphaeriaceae family are known to cause Bot gummosis on many woody plants worldwide. To identify pathogens associated with Bot gummosis on citrus in California, scion and rootstock samples were collected in 2010 and 2011 from five citrus-growing counties in California. Symptoms observed on citrus included branch cankers, dieback, and gumming. Various fungal species were recovered from necrotic tissues of branch canker and rootstock samples. Species were identified morphologically and by phylogenetic comparison as 'Eureka' lemon, 'Valencia', 'Washington Navel', 'Fukumoto', grapefruit, 'Satsuma', and 'Meyer' lemon. Species were identified morphologically and by phylogenetic comparison of the complete sequence of the internal transcribed spacer regions, ß-tubulin gene, and elongation factor α-1 genes with those of other species in GenBank. A consensus-unrooted most parsimonious tree resulting from multigene phylogenetic analysis showed the existence of three major clades in the Botryosphaeriaceae family. In total, 74 isolates were identified belonging to the Botryosphaeriaceae family, with Neofusicoccum spp., Dothiorella spp., Diplodia spp., (teleomorph Botryosphaeria), Lasiodiplodia spp., and Neoscytalidium dimidiatum (teleomorphs unknown) accounting for 39, 25, 23, 10, and 3% of the total, respectively. On inoculated Eureka lemon shoots, lesion length was significantly different (P < 0.05) among 14 isolates recovered from portions of cankered tissues of the original trees. Lesion lengths were significantly longer (P < 0.05) for shoots inoculated with isolates of Neofusicoccum luteum and shorter for shoots inoculated with isolates of Dothiorella viticola (P < 0.05) than those of other species. Identifying the distribution and occurrence of these fungal pathogens associated with Bot gummosis is useful for management applications during occasional outbreaks in California.

First Report of Spencermartinsia viticola, Neofusicoccum australe, and N. parvum Causing Branch Canker of Citrus in California.

Dothiorella gummosis and canker on citrus is generally viewed as a minor disease but can result in serious decline of trees. Symptoms, mostly found on branches, include grayish-to-brown cast on cankered bark, which can extend into the xylem. Dothiorella gummosis was earlier believed to be caused by Dothiorella gregaria (2). In a continuing survey on citrus in six California counties (Fresno, Riverside, San Diego, San Luis Obispo, Tulare, and Ventura) in 2010, branch cankers were collected. Small pieces of symptomatic tissues were plated onto potato dextrose agar amended with 0.01% tetracycline (PDA-tet) and incubated at 25°C for 4 days. Fungi most frequently isolated were initially identified as Botryosphaeriaceae based on morphological characters (1,3). Total genomic DNA was PCR amplified with primers Bt2a/2b for the ß-tubulin (BT); EF1-728F/986R for the elongation factor α-1 (EF); and ITS4/5 for the internal transcribed spacer ITS1-5.8S-ITS2 regions (3). Sequences were compared in a BLAST search. Spencermartinsia viticola UCP105 was isolated from cv. Parent Washington on Sour Orange rootstock in Tulare County, Neofusicoccum australe UCR1110 from cv. Satsuma in Riverside County, and N. parvum UCR1166 from cv. Meyer Lemon on Volkameriana rootstock in Ventura County. Sequences of UCP105, UCR1110, and UCR1166 have been deposited in GenBank under Accession Nos. JF271766, JF271776, and JF271780 for BT; JF271784, JF271793, and JF271796 for EF; and JF271748, JF271758, and JF271762 for the ITS regions. The sequences matched with isolates in GenBank as follows: ITS region of strain UCP105-98% match with Accession Nos. AY905556-8; BT of strain UCR1110-99% with GU251879-80; and EF of strain UCR1166-98% with GU251238. Pathogenicity tests were conducted by inoculating green shoots of healthy citrus trees similar to cultivar/rootstock from which each isolate was obtained. Fresh wounds were made on 1-year-old citrus shoots with a 3-mm cork borer, and the freshly wounded surfaces were inoculated with 3-mm mycelial plugs from 5-day-old cultures on PDA-tet. Control shoots were inoculated with sterile agar plugs and each treatment had 10 replicates. Inoculated wounds and shoot ends were covered with petroleum jelly and wrapped with Parafilm to prevent desiccation. Shoots were incubated at 25°C in moist chambers for 4 weeks. Lesions were observed on all inoculated shoots except for the control. Mean lesion lengths were 6.4, 7.0, and 6.9 cm for UCP105, UCR1110, and UCR1166, respectively, which were significantly (P = 0.05) different from the control (0.8 cm). The three isolates were reisolated from symptomatic tissues of inoculated shoots to confirm their pathogenicity. This test was repeated and similar results were obtained. Results indicate that there are multiple species in the Botryosphaeriaceae family causing symptoms on citrus that were previously believed to be caused by D. gregaria. To our knowledge, this is the first report of S. viticola, N. australe, and N. parvum on citrus in California. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) V. McDonald et al. Plant Dis. 93:967, 2009. (3) B. Slippers et al. Mycologia 96:83, 2004.

First Report of Eutypella spp. Associated with Branch Canker of Citrus in California.

Eutypella is one of the few genera in the Diatrypaceae considered plant pathogens (1). In California, E. vitis and other members of the Diatrypaceae cause branch and trunk canker on grapevine (3,4). Eutypella spp. have not previously been documented as pathogens of citrus. In a 2010 survey on citrus branch canker and dieback in six citrus-growing counties of California, four isolates of Eutypella species were detected in Riverside and San Diego counties. Canker symptoms included dieback and bark cracking, and cuts made through symptomatic trees showed that the cankers were expanding through the center of the tree. Branch samples were collected from 10 trees per orchard and 5 to 10 orchards per county (102 trees for two counties). Pieces of symptomatic tissue (1 to 2 mm2) were plated onto potato dextrose agar amended with 0.01% tetracycline (PDA-tet) and incubated at 25°C for 4 days. All isolates were identified by morphological and molecular characteristics. PCR of isolates was performed in a thermal cycler using two primer pairs, ITS4/5 and Bt2a/2b for amplifying the internal transcribed spacer (ITS1), 5.8S, and ITS2 region and the ß-tubulin gene, respectively (2,3). PCR products were sequenced at the University of California, Riverside Genomics Core and the sequences compared in a BLAST search. Four isolates identified as Eutypella spp. included two (UCR1088 and UCR1101) from San Diego County and two (UCR1148 and UCR1149) from the Riverside County samples. The sequences were deposited in GenBank (HQ880579, JF758610, HQ880581, and HQ880582 and HQ880583, JF758611, HQ880585, and HQ880586 for the ITS regions and ß-tubulin gene, respectively. ITS sequences for UCR1088 and UCR1101 had 98 and 100% match, respectively, to Eutypella spp. ITS sequences in GenBank (GQ293959 to GQ293961), while UCR1148 and UCR1149 matched 99% (GQ293956 to GQ293958). On the basis of morphological characteristics, UCR1088 and UCR1101 were similar to Eutypella spp. group 1, while UCR1148 and UCR1149 were similar to Eutypella spp. group 3 (4). Pathogenicity tests were conducted with all four isolates on detached shoots from healthy citrus trees of the same cultivar/rootstock from which each isolate was obtained. One wound per shoot was made on 1-year-old, green, detached shoots using a 3-mm-diameter cork borer and the wounded surfaces were inoculated with 3-mm-diameter mycelial plugs of 5-day-old cultures of each isolate growing on PDA-tet. Inoculated wounds and shoot ends were covered with petroleum jelly and wrapped with Parafilm (3). Control shoots were inoculated with sterile agar plugs. There were 10 inoculated shoots per isolate and noninoculated control treatment. Shoots were incubated at 25°C in moist chambers for 6 weeks. Lesions similar to those on the original infected shoots were observed on all inoculated shoots except the control treatment. Reisolation and identification of fungi from inoculated and control shoots were done using methods described above. Inoculated isolates were recovered from 100% of inoculated shoots but none was recovered from noninoculated shoots, indicating association of Eutypella spp. with citrus branch canker. To our knowledge, this is the first report of Eutypella spp. associated with cankers on citrus in California. References: (1) B. Piskur et al. Plant Dis. 91:1579, 2007. (2) B. Slippers et al. Mycologia 96:83, 2004. (3) F. P. Trouillas and W. D. Gubler. Plant Dis. 94:867, 2010. (4) F. P. Trouillas et al. Mycologia 102:319, 2010.

Plant growth-promoting rhizobacteria allow reduced application rates of chemical fertilizers.

The search for microorganisms that improve soil fertility and enhance plant nutrition has continued to attract attention due to the increasing cost of fertilizers and some of their negative environmental impacts. The objectives of this greenhouse study with tomato were to determine (1) if reduced rates of inorganic fertilizer coupled with microbial inoculants will produce plant growth, yield, and nutrient uptake levels equivalent to those with full rates of the fertilizer and (2) the minimum level to which fertilizer could be reduced when inoculants were used. The microbial inoculants used in the study were a mixture of plant growth-promoting rhizobacteria (PGPR) strains Bacillus amyloliquefaciens IN937a and Bacillus pumilus T4, a formulated PGPR product, and the arbuscular mycorrhiza fungus (AMF), Glomus intraradices. Results showed that supplementing 75% of the recommended fertilizer rate with inoculants produced plant growth, yield, and nutrient (nitrogen and phosphorus) uptake that were statistically equivalent to the full fertilizer rate without inoculants. When inoculants were used with rates of fertilizer below 75% of the recommended rate, the beneficial effects were usually not consistent; however, inoculation with the mixture of PGPR and AMF at 70% fertility consistently produced the same yield as the full fertility rate without inoculants. Without inoculants, use of fertilizer rates lower than the recommended resulted in significantly less plant growth, yield, and nutrient uptake or inconsistent impacts. The results suggest that PGPR-based inoculants can be used and should be further evaluated as components of integrated nutrient management strategies.

Enhanced plant nutrient use efficiency with PGPR and AMF in an integrated nutrient management system.

A 3 year field study was conducted with field corn from 2005 to 2007 to test the hypothesis that microbial inoculants that increase plant growth and yield can enhance nutrient uptake, and thereby remove more nutrients, especially N, P, and K from the field as part of an integrated nutrient management system. The field trial evaluated microbial inoculants, which include a commercially available plant growth-promoting rhizobacteria (PGPR), arbuscular mycorrhiza fungi (AMF), and their combination across 2 tillage systems (no-till and conventional till) and 2 fertilization regimes (poultry litter and ammonium nitrate). Data were collected on plant height, yield (dry mass of ears and silage), and nutrient content of corn grain and silage. In addition, nutrient content of soil was determined, and bioavailability of soil nutrient was measured with plant root simulator probes. Results showed that inoculants promoted plant growth and yield. For example, grain yields (kg.ha(-1)) in 2007 for inoculants were 7717 for AMF, 7260 for PGPR+AMF, 7313 for PGPR, 5725 for the control group, and for fertilizer were 7470 for poultry litter and 6537 for NH4NO3. Nitrogen content per gram of grain tissues was significantly enhanced in 2006 by inoculant, fertilizer, and their interactions. Significantly higher amounts of N, P, and K were removed from the plots with inoculants, based on total nutrient content of grain per plot. These results supported the overall hypothesis and indicate that application of inoculants can lead to reduction in the build up of N, P, and K in agricultural soils. Further studies should be conducted to combine microbial inoculants with reduced rates of fertilizer.

Comparison of plant growth-promotion with Pseudomonas aeruginosa and Bacillus subtilis in three vegetables / Comparação da promoção de crescimento de plantas por Pseudomonas aeruginosa e Bacillus subtilis em três vegetais

Our objective was to compare some plant growth promoting rhizobacteria (PGPR) properties of Bacillus subtilis and Pseudomonas aeruginosa as representatives of their two genera. Solanum lycopersicum L. (tomato), Abelmoschus esculentus (okra), and Amaranthus sp. (African spinach) were inoculated with the bacterial cultures. At 60 days after planting, dry biomass for plants treated with B. subtilis and P. aeruginosa increased 31 percent for tomato, 36 percent and 29 percent for okra, and 83 percent and 40 percent for African spinach respectively over the non-bacterized control. Considering all the parameters tested, there were similarities but no significant difference at P < 0.05 between the overall performances of the two organisms.

Nosso objetivo foi comparar as propriedades PGPR (rizobactérias promotoras de crescimento de plantas) de Bacillus subtilis e Pseudomonas aeruginosa. Solanum licopersicum (tomate), Asbelmoschus esculentus (ocra) e Amaranthus sp (espinafre africano) foram inoculados com as culturas bacterianas. Após 60 dias de plantio, a biomassa seca das plantas tratadas com B.subtilis e P. aeruginosa aumentou 31 por cento para o tomate, 36 por cento e 29 por cento para ocra, e 83 por cento e 40 por cento para espinafre africano, respectivamente, em comparação com o controle não inoculado. Considerando os parâmetros testados, o desempenho dos dois microrganismos foi similar, sem diferença estatisticamente significativa (p< 0,05).

Comparison of plant growth-promotion with Pseudomonas aeruginosa and Bacillus subtilis in three vegetables.

Our objective was to compare some plant growth promoting rhizobacteria (PGPR) properties of Bacillus subtilis and Pseudomonas aeruginosa as representatives of their two genera. Solanum lycopersicum L. (tomato), Abelmoschus esculentus (okra), and Amaranthus sp. (African spinach) were inoculated with the bacterial cultures. At 60 days after planting, dry biomass for plants treated with B. subtilis and P. aeruginosa increased 31% for tomato, 36% and 29% for okra, and 83% and 40% for African spinach respectively over the non-bacterized control. Considering all the parameters tested, there were similarities but no significant difference at P < 0.05 between the overall performances of the two organisms.