Symptoms of the disease in the field
The diseased palms were individually scattered in the field, and the visual percentage of infected or dead palms was 3–5%. Symptomatology of the disease in the field included wilting of the youngest leaves, first manifested as a change in the leaf color to a paler green that progressed to a green brown and eventually to light brown as the leaves became desiccated and wilted (Figs. 1 and 2), and when making a cross section at the base of the diseased palm, the bases of the petioles show brown to dark brown soft tissues with a foul smell (Fig. 2). Progressive separation of the petiole bases of diseased palms showed tan-colored necrotic lesions, with brown margins. The surface of some lesions was covered by layers of white mycelia (Fig. 2). At the late stages of the disease, pale leaves completely died, buds were totally rotted, and leaf petioles disintegrated leaving a black cavity in the base of the dead palm (Fig. 3). Making a transverse section of a diseased tree at the petiole showed a brown discoloration and rot at the base and terminal bud tissues (Fig. 3). Bud (heart) rot in palm trees are caused by many species of Phytophthora in the world. P. nicotianae has been reported to infect blue Mediterranean fan palm (Chamaerops humilis var. argenta), causing basal leaf rot in Iran (Nazerian and Mirabolfathi 2013), in North America (Bomberger et al. 2016), and in Italy (Faedda et al. 2011). Therefore, bamboo palm, Chamaedorea erumpens; reed palm, C. seifrizii; Macarthur palm, Ptychosperma macarthurii; red sealing wax palm, Cyrtosta chysrenda; thatch palm, Thrinax sp.; and Mexican fan palm, W. robusta have been reported infected by P. nicotianae with stem, leaf, and root rots (Elliott et al. 2004). P. palmivora attacked W. robusta and W. filifera, causing one bud rot.
Pathogenicity test
All the three Phytophthora isolates (Phyto1, Phyto2, and Phyto3) were highly pathogenic to W. robusta young trees. Infected plants were pale, weathered, and growing poorly compared to inoculated plants 3 weeks after inoculation. The inoculated plants were almost dead at the fifth week compared to inoculated check plants (Fig. 4). When infected trees were longitudinally split by a sharp knife after 5–6 weeks from inoculation, the terminal bud and petiole bases were totally decayed showing brown to black rotted tissues (Fig. 5). Isolation from the dead and diseased tissues of the artificially inoculated trees revealed that all the tissues were infected with the same pathogen that was used for their inoculation.
Microscopic examination
The three isolated fungi were identical in their morphological characteristics. The pathogen was identified as P. nicotianae that was characterized by thick and branched hyphal growth (5–7.1 μm). Zoosporangia (30.36–43.97 μm) were formed on the mycelium 3–4 days after an incubation at 28 °C (Fig. 6a). Thickly walled sporangiospores (59.18–68.42 μm) were formed extensively on the mycelium in 7–15-day-old cultures (Fig. 6b). Sporangia were formed 3–4 days after an incubation at 28 °C and an extensive formation of chlamidospores was observed in culture plates of 7–15 days old. The results presented above are characteristics of P. nicotianae based on previous studies reported by Stamps et al. (1990) and Faedda et al. (2011). Traditionally, Phytophthora species taxonomic identification has been based upon microscopic examination of morphological characters and cultural criteria (Stamps et al. 1990).
P. nicotianae causes seedling blight of golden palm and also attacks California fan palm, causing trunk and collar rots. The disease appears to begin from wounds caused by leaf removal at or near the soil line. Although this disease can be reproduced by inoculations of the lower trunk, the bud and the entire stem are eventually rotted on some plants. Thus, P. nicotianae should also be considered as a possible cause of bud rots.
Molecular characterization of P. nicotianae isolates
A band of about 900 bp was amplified using total DNA from the three isolates (phyto1, phyto2, and phyto3) of P. nicotianae using the universal ITS4/ITS5 primers (Fig. 7). The three PCR products were sequenced obtaining for phyto1, phyto2, and phyto3 881, 843, and 843 bp, respectively. ITS region was conventionally used for the molecular identification of plant pathogenic fungi (Durán et al. 2010). The fungal ITS region varies between approximately 450 and 750 bp in length and consists of the variable spacers ITS1 and ITS2 and the intercalary 5.8S gene (Schoch et al. 2012). The ITS region sequences of P. nicotianae isolates were compared by other ITS sequences of Phytophthora species reported in GenBank (www.ncbi.nlm.nih.gov). The phylogenetic results (Fig. 8) revealed that the Phytophthora isolates formed two clades (I and II). Clade I includes the three P. nicotianae isolates from Qassim region and all P. nicotianae and P. parasitica isolates. However, the three P. nicotianae isolates from Qassim region have been separated and formed subclade B with P. nicotianae isolate 2013CAP2002 (KJ549640) from the USA and P. nicotianae strain IIFT 155 (GU073389) from Cuba with 100% of the identity confirming genetic similarity of isolates (Fig. 8). P. palmivora strains were distanced from other Phytophthora isolates by bootstrap 100% and grouped together in clade II. The Qassim P. nicotianae isolates had more than (99.6%) nucleotide identity with P. nicotianae and P. parasitica isolates, whereas the identities were less than (89.3%) with all P. palmivora strains. An analysis of nucleotide sequences of internal transcribed spacer (ITS) regions of rDNA has been used to differentiate Phytophthora species (Cooke et al. 2000, Förster et al. 2000, and Meenupriya and Thangaraj 2011). Also, the ITS could be successfully used for discriminating closely related species of a broad range of fungi (Schoch et al. 2012).
Antifungal activities
As shown in Fig. 9, the growth of P. nicotianae (Phyto1 isolate) was more inhibited by using the bacterial strain L. enzymogenes BB14 than in the control treatment. The tested bacterial isolate exhibited stronger inhibitory effects on mycelial growth of P. nicotianae after 7 days, nearly 100% inhibition percentage. The potential biological control for plant diseases has been recently reported, using Lysobacter species (Folman et al. 2003 and Sullivan et al. 2003). Li et al. (2008) reported that L. enzymogenes strain C3 is a biological control agent that has a strong antagonist effect against several fungal pathogens. L. enzymogenes has been reported to suppress soil-borne diseases, such as Rhizoctonia solani that caused brown patch in turf grass (Giesler and Yuen 1998). Moreover, L. enzymogenes has been cited to control foliar diseases, for example, Bipolaris sorokiniana causal agent of leaf spot of tall fescue (Zhang and Yuen 1999), Uromyces appendiculatus causal agent of bean rust (Yuen et al. 2001), and Fusarium head blight of wheat (Jochum et al. 2006).
In this study, the bacterial isolate L. enzymogenes BB14 was more active in chitin hydrolysis as shown in Fig. 10 which gives it more antifungal activity against P. nicotianae. Li et al. (2008) mentioned that the antifungal activity of L. enzymogenes was attributed in part to lytic enzymes. Many extracellular enzymes that contribute to biocontrol activity, including multiple forms of β-1, 3-glucanases, and chitinases, are produced by L. enzymogenes as reported by Zhang et al. (2001) and Palumbo et al. (2005).